减数分裂特异性远端粘连位点与着丝粒分离-动脉网

Meiosis-specific distal cohesion site decoupled from the kinetochore

Nature 等信源发布 2025-03-03 22:31

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Abstract

摘要

Primary constriction of the M-phase chromosome serves as a marker for the kinetochore position. Underlying this observation is the concept that the kinetochore is spatially linked with the pericentromere where sister-chromatids are cohered. Here, we find an unconventional chromatid-cohesion pattern in .

M期染色体的主缢痕是动粒位置的标记。这一观察结果的背后是动粒与姐妹染色单体相凝聚的着丝粒周围区域在空间上相关联的概念。在此，我们发现了一种非传统的染色单体凝聚模式。

Peromyscus

鹿鼠属

oocytes, with sister chromatids cohered at a chromosome end, spatially separated from the kinetochore. This distal locus enriches cohesin protectors specifically during meiosis, and chromosomes with this additional cohesion site exhibit enhanced cohesin protection at anaphase I compared to those without it, implying an adaptive evolution to ensure cohesion during meiosis.

卵母细胞中，姐妹染色单体在染色体末端保持凝聚，与着丝粒空间分离。这个远端位点在减数分裂期间特异性地富集了黏连蛋白保护因子，与没有这一额外凝聚位点的染色体相比，具有该位点的染色体在减数分裂I期表现出更强的黏连蛋白保护作用，这暗示了一种确保减数分裂期间凝聚的适应性进化。

The distal locus corresponds to an additional centromeric satellite block, located far from the satellite block building the kinetochore. Analyses on three .

远端位点对应于一个额外的着丝粒卫星区块，位于远离构建动粒的卫星区块的位置。对三个进行了分析。

Peromyscus

鹿鼠属

species reveal that the internal satellite consistently assembles the kinetochore in mitosis and meiosis, whereas the distal satellite selectively enriches cohesin protectors in meiosis to promote cohesion. Our study demonstrates that cohesion regulation is flexible, controlling chromosome segregation in a cell-type dependent manner..

物种揭示了内部卫星在有丝分裂和减数分裂中始终组装着丝粒，而远端卫星在减数分裂中选择性地富集黏连蛋白保护因子以促进黏连。我们的研究表明，黏连调控具有灵活性，以细胞类型依赖的方式控制染色体分离。

Introduction

简介

Accurate chromosome segregation during mitosis and meiosis is crucial for maintaining genomic stability and ensuring the faithful inheritance of genetic material across generations. There are at least two fundamental and evolutionarily conserved features of M-phase chromosomes to ensure faithful segregation: (1) the assembly of the kinetochore to interact with the spindle apparatus and (2) the cohesion of sister chromatids to ensure bi-orientation of the chromosome.

有丝分裂和减数分裂过程中准确的染色体分离，对于维持基因组稳定性以及确保遗传物质在世代间的忠实传递至关重要。M期染色体至少具有两个基本且在进化上保守的特征，以确保忠实的分离：(1) 动粒的组装以与纺锤体相互作用；(2) 姐妹染色单体的粘连以确保染色体的双取向。

，

. The kinetochore position and the sister-chromatid cohesion site are spatially linked and located on centromeric satellite DNA in many species. Indeed, the primary constriction site, where the sisters are most tightly cohered, is a classic indicator for the centromere/kinetochore position to determine the karyotype of each species, first described by Walter Flemming in 1882.

着丝粒的位置和姐妹染色单体的粘连位点在许多物种中是空间上相连的，并位于着丝粒卫星DNA上。事实上，姐妹染色单体最为紧密粘连的主缢痕位置是确定每个物种核型的经典指标，这一特征最早由瓦尔特·弗莱明于1882年描述。

，

. At a molecular level, chromosome cohesion is mediated by the cohesin complex, which initially loads along the chromosome axis. Upon mitotic entry, cohesin on the chromosome arm is removed by the prophase pathway, whereas pericentromeric cohesin is protected until anaphase onset where the Separase-mediated cleavage takes place, allowing chromosome segregation.

在分子水平上，染色体的凝聚力由黏连蛋白复合体介导，该复合体最初沿着染色体轴装载。在有丝分裂进入时，染色体臂上的黏连蛋白通过前中期途径被移除，而着丝粒周围的黏连蛋白则受到保护，直到后期开始时发生分离酶介导的切割，从而允许染色体分离。

Kinetochores play a role in this cohesin protection at the pericentromere by recruiting Chromosomal Passenger Complex (CPC) and the Shugoshin (SGO)-PP2A complex to the pericentromere.

着丝粒通过将染色体乘客复合体（CPC）和护送蛋白（SGO）-PP2A复合体招募到着丝粒周围区域，在该区域的黏连蛋白保护中发挥作用。

，

. On the other hand, CPC at the pericentromere facilitates kinetochore assembly at the centromere in several organisms

另一方面，在着丝粒周围区域的CPC有助于多种生物在着丝粒处的动粒组装。

，

. Therefore, there are multiple molecular links between the kinetochore and the pericentromere to ensure proper chromosome segregation.

因此，着丝粒和近着丝粒之间存在多种分子联系，以确保染色体的正确分离。

Despite the essential and conserved role of centromeres in chromosome segregation, it paradoxically represents the most rapidly evolving part of the genome

尽管着丝粒在染色体分离中起着关键且保守的作用，但它却矛盾地代表了基因组中进化最快的那部分。

，

. The functional consequences of rapid centromere evolution are largely unknown. Particularly, the impact of centromere evolution on pericentromere specification and functions have not been investigated at a molecular level. The

快速着丝粒进化的功能后果在很大程度上是未知的。特别是，着丝粒进化对近着丝粒特异性和功能的影响尚未在分子水平上得到研究。

Peromyscus

鹿鼠属

mouse is an ideal system to tackle this question because of their rapid centromere evolution in both size and position, driving karyotypic diversity across the

由于小鼠的着丝粒在大小和位置上快速进化，驱动了整个核型的多样性，因此它是解决这个问题的理想系统。

Peromyscus

鹿鼠属

genus

属

，

。

Peromyscus

鹿鼠属

satellite (PMsat) are satellite repeats that locate at the (peri)centromere region in various

卫星（PMsat）是位于各种（近）着丝粒区域的卫星重复序列。

Peromyscus

鹿鼠属

mouse species

鼠标种类

，

. Previous studies revealed that PMsat is present at the (peri)centromeric region of all chromosomes. Interestingly, PMsat is located also at non-centromeric regions proximal to telomeres (hereafter telomeric PMsat) in several chromosomes (e.g., chromosome 18, 21, and 22 in

以往的研究表明，PMsat存在于所有染色体的（近）着丝粒区域。有趣的是，在多个染色体中，PMsat也位于靠近端粒的非着丝粒区域（以下简称端粒PMsat）（例如，第18、21和22号染色体）。

Peromyscus maniculatus

北美鹿鼠

, hereafter referred to as dual PMsat chromosomes) (Fig.

，以下简称双PMsat染色体）（图。

). We took advantage of these naturally occurring chromosomes harboring two blocks of centromeric satellites to investigate the impact of centromere evolution on (peri)centromere specification.

）。我们利用这些含有两个着丝粒卫星区块的天然染色体，研究着丝粒进化对（近）着丝粒特异性的影响。

Fig. 1: Kinetochores assemble exclusively at internal PMsat on dual PMsat chromosomes.

图1：动粒仅在双PMsat染色体的内部PMsat上组装。

Phylogenetic tree of mouse species in the

小鼠物种的系统发育树

Mus

鼠标

，

Rattus

鼠属

, and

，以及

Peromyscus

鹿鼠属

genus. For each

属。对于每个

Peromyscus

鹿鼠属

species, the chromosomal distribution of PMsat is shown based on Smalec et al.

物种，PMsat 的染色体分布基于 Smalec 等人的研究展示。

。

Peromyscus maniculatus

鹿鼠

and

和

Peromyscus polionotus

北美鹿鼠

but not

但是不

Peromyscus californicus

加州白足鼠

carry chromosomes with two PMsat blocks.

携带具有两个PMsat块的染色体。

CENP-A enrichment at PMsat regions. CENP-A and IgG enrichment on PMsat sequences is provided as ratio of ChIP signal over the input (left). IGV snapshots of CENP-A enrichment (ratio over input) at PMsat regions on two chromosomes (right).

CENP-A在PMsat区域的富集。CENP-A和IgG在PMsat序列上的富集以ChIP信号与输入的比值表示（左）。两个染色体上PMsat区域CENP-A富集（相对于输入的比值）的IGV快照（右）。

, Metaphase chromosome spread using

，使用中期染色体扩散

P. maniculatus

mitotic cells (ovarian granulosa cells) were stained for PMsat (Oligopaint) and a kinetochore marker, HEC1.

有丝分裂细胞（卵巢颗粒细胞）被染色以标记PMsat（Oligopaint）和动粒标记物HEC1。

P. maniculatus

鹿鼠属

meiosis II oocytes expressing dCas9-EGFP and gRNA targeting PMsat were used for chromosome spread and stained for HEC1. The proportion of chromosomes that assemble kinetochores at internal PMsat and telomeric PMsat was quantified;

表达 dCas9-EGFP 和靶向 PMsat 的 gRNA 的减数分裂 II 卵母细胞用于染色体铺展并用 HEC1 染色。量化了在内部 PMsat 和端粒 PMsat 上组装动粒的染色体比例；

= 24 and 56 cells from at least three independent experiments were examined for mitosis (

= 24 和 56 个细胞来自至少三个独立实验，检查了有丝分裂 (

) and meiosis II (

）和减数第二次分裂（

), respectively. The images are maximum projections showing all the chromosomes (left) and optical sections to show individual chromosomes (right); asterisks denote the chromosomal location of internal PMsat (orange) and telomeric PMsat (yellow) on dual PMsat chromosomes; scale bars, 5 µm.

），分别为最大投影显示所有染色体（左）和光学切片显示单个染色体（右）；星号表示双PMsat染色体上内部PMsat（橙色）和端粒PMsat（黄色）的染色体位置；比例尺，5微米。

Full size image

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Results

结果

PMsat is the centromeric satellite of

PMsat 是着丝粒卫星

Peromyscus maniculatus

北美鹿鼠

We first confirmed that PMsat is the centromeric satellite for

我们首先确认 PMsat 是着丝粒卫星

Peromyscus

鹿鼠属

mice. Centromeres enrich specialized nucleosomes containing the histone H3 variant, CENP-A, which defines the kinetochore assembly site

小鼠。着丝粒富含包含组蛋白H3变体CENP-A的特殊核小体，其定义了动粒组装位点。

，

. We enriched CENP-A chromatin from

我们从CENP-A染色质中富集了

Peromyscus maniculatus

鹿鼠

granulosa cells by chromatin immunoprecipitation (ChIP). High-throughput sequencing and analysis revealed that CENP-A is enriched at regions containing the PMsat sequence (Fig.

通过染色质免疫沉淀（ChIP）技术检测颗粒细胞。高通量测序和分析显示，CENP-A在含有PMsat序列的区域富集（图。

and Supplementary Fig.

和补充图。

). We observed a strong association of genomic regions enriched for CENP-A binding and containing the PMsat sequence (Supplementary Fig.

我们观察到富含CENP-A结合并包含PMsat序列的基因组区域之间存在很强的关联（补充图）。

1c, d

). Furthermore, de novo motif discovery analysis in the sequences underlying CENP-A peaks confirmed the presence of the PMsat consensus sequence (Supplementary Fig.

）。此外，对CENP-A峰下层序列进行的从头 motif 发现分析确认了PMsat共识序列的存在（补充图。

), demonstrating that PMsat is the primary centromeric satellite in

），这表明PMsat 是主要的着丝粒卫星在

Peromyscus maniculatus

鹿鼠

。

Internal PMsat builds the kinetochore in mitosis and meiosis

内部PMsat在有丝分裂和减数分裂中构建着丝粒。

We next tested how the centromere position is specified when a chromosome harbors one or two centromeric satellite blocks. HEC1, a major outer kinetochore component, co-localized with PMsat and CENP-A in all standard chromosomes with a single PMsat locus in mitotic chromosome spreads (Fig.

我们接下来测试了当染色体包含一个或两个着丝粒卫星区块时，着丝粒位置是如何确定的。HEC1 是主要的外层动粒成分，与 PMsat 和 CENP-A 在所有具有单个 PMsat 位点的标准染色体的有丝分裂染色体铺展中共定位（图）。

and Supplementary Fig.

和补充图。

, standard), confirming that PMsat is indeed the centromeric satellite of this species. Interestingly, kinetochores were always and solely assembled on internal PMsat (instead of telomeric PMsat) on all dual PMsat chromosomes (Fig.

，标准），证实 PMsat 确实是该物种的着丝粒卫星。有趣的是，动粒总是且仅组装在所有双 PMsat 染色体的内部 PMsat 上（而非端粒 PMsat）（图。

and Supplementary Fig.

和补充图。

), implying (1) a selective pressure to utilize the internal centromeric satellite to form the kinetochore and (2) a silencing of telomeric PMsat to avoid the formation of dicentric chromosomes. Since somatic cells and oocytes can have distinct regulation of centromeric chromatin

)，这意味着（1）选择性压力利用内部着丝粒卫星形成动粒，以及（2）沉默端粒PMsat以避免形成双着丝粒染色体。由于体细胞和卵母细胞可能对着丝粒染色质有不同调控，

, the kinetochore position was also analyzed in meiosis I and II oocytes. Similar to mitotic cells, oocytes assembled their kinetochores at internal PMsat, demonstrating stable specification of the centromere position (Fig.

，还分析了减数分裂I和II卵母细胞中的动粒位置。与有丝分裂细胞相似，卵母细胞在内部PMsat处组装它们的动粒，表明着丝粒位置的稳定特化（图。

1天

and Supplementary Fig.

和补充图。

2a–d

). This centromere specification pattern was conserved in another species,

）。这种着丝粒特异性模式在另一物种中也是保守的，

Peromyscus polionotus

旧地松鼠

, which became evolutionary separated approximately 100,000 years ago

，它们在大约10万年前进化分离

and has different chromosomes with dual PMsat blocks (i.e., chromosome 16, 18, and 19) (Fig.

并且具有带有双重PMsat块的不同染色体（即，染色体16、18和19）（图。

and Supplementary Fig.

和补充图。

3a,b

). Collectively, these results show that the kinetochore position is stably maintained at the internal centromeric satellite block across different tissues and species.

这些结果共同表明，着丝粒位置在不同组织和物种中都能稳定地保持在内部着丝粒卫星区块。

Telomeric PMsat is the major cohesion site in oocyte meiosis

端粒PMsat是卵母细胞减数分裂中的主要粘连位点

Compared to centromere specification, how the pericentromere is specified is less studied mainly due the general assumption that kinetochore and the pericentromeric cohesion site are spatially linked. Chromosomes with a centromere in the mid-way (i.e., metacentric chromosomes) generally show the characteristic X-shape morphology during M-phase because sister chromatids are tied together in the mid-way while the chromosome arms are separated (Supplementary Fig. .

与着丝粒特化相比，由于普遍认为动粒和着丝粒周围的凝聚位点在空间上是相连的，因此对如何特化着丝粒周围区域的研究较少。具有中间着丝粒（即，中着丝粒染色体）的染色体通常在M期显示出特征性的X形形态，因为姐妹染色单体在中间被绑在一起，而染色体臂则分开（补充图）。

). On the other hand, telocentric chromosomes with their centromeres at the chromosome end show V-shape morphology because they are cohered at one end of the chromosome where the centromere resides. These observations established the concept that the major cohesion site is spatially linked with the centromere regardless of the centromere position.

）。另一方面，由于着丝粒位于染色体的一端，端着丝粒染色体呈现出V形形态，因为它们在着丝粒所在的染色体一端保持凝聚。这些观察结果确立了一个概念，即主要的凝聚位点在空间上与着丝粒相关联，而无论着丝粒的位置如何。

The unique centromere organization of .

独特的着丝粒组织。

Peromyscus

鹿鼠属

chromosomes prompted us to revisit this dogma and test the impact of dual centromeric satellite blocks on the sister-chromatid cohesion pattern. We examined the cohesion site of dual PMsat chromosomes in whole-mount cells where spindle microtubule pulling forces are present (in contrast to chromosome spreads in Fig. .

染色体促使我们重新审视这一教条，并测试双重着丝粒卫星区块对姐妹染色单体凝聚模式的影响。我们在整体细胞中检查了具有双重PMsat染色体的凝聚位点，这些细胞中存在纺锤体微管拉力（与图中的染色体扩散相对比）。

1c, d

) (Fig.

) （图。

). In mitosis, sister chromatids were tightly cohered at internal PMsat (i.e., a single PMsat peak in the line scan) whereas chromatids were separated at telomeric PMsat (i.e., two separate PMsat peaks) (Fig.

在有丝分裂中，姐妹染色单体在内部的PMsat处紧密相连（即，在线扫描中呈现单一的PMsat峰），而在端粒的PMsat处染色单体分离（即，两个独立的PMsat峰）（图。

, mitosis). Therefore, internal PMsat assembles the kinetochore and also serves as the major cohesion site for dual PMsat chromosomes similar to standard chromosomes. In meiosis II, sister kinetochores were more separated compared to mitosis as previously reported

，有丝分裂）。因此，内部的PMsat组装着丝粒，也像标准染色体一样，作为双PMsat染色体的主要粘连位点。在减数分裂II中，姐妹着丝粒比有丝分裂时分离得更开，正如之前所报道的那样。

，

, particularly for dual PMsat chromosomes (Fig.

，特别是对于双PMsat染色体（图。

, meiosis II, Supplementary Fig.

，减数分裂II，补充图。

). We found that dual PMsat chromosomes were consistently cohered at telomeric PMsat, showing a single PMsat peak in the line scan (Fig.

）。我们发现双重PMsat染色体在端粒PMsat处始终保持一致，线扫描显示单个PMsat峰（图。

, meiosis II dual, and Supplementary Fig.

，减数分裂II双重，以及补充图。

). These results imply that chromosomes with two centromeric satellite blocks can switch over the major cohesion site to a distal centromeric satellite block during female meiosis while the kinetochore position remains stable.

）。这些结果表明，在雌性减数分裂过程中，具有两个着丝粒卫星区块的染色体可以将主要的粘连位点切换到远端的着丝粒卫星区块，而动粒的位置保持稳定。

Fig. 2: Telomeric PMsat acts as the primary cohesion site in oocytes.

图2：端粒PMsat在卵母细胞中充当主要的粘连位点。

P. maniculatus

mitotic cells (ovarian granulosa cells) and meiosis II oocytes expressing dCas9-mCherry with gRNA targeting PMsat were fixed and stained for HEC1. For the mitotic cells, PMsat was labeled by Oligopaint. Line scans of the signal intensities of HEC1 and PMsat across the PMsat loci were performed;

表达dCas9-mCherry且gRNA靶向PMsat的有丝分裂细胞（卵巢颗粒细胞）和减数分裂II期卵母细胞被固定并染色以检测HEC1。对于有丝分裂细胞，PMsat通过Oligopaint标记。对PMsat位点上HEC1和PMsat信号强度进行了线扫描。

= 13 and 46 cells from three and 11 independent experiments were analyzed for mitosis and meiosis II, respectively; lines represent the mean intensities; error bars, SD.

= 13，分别从三次和十一次独立实验中分析了有丝分裂和减数分裂II的46个细胞；线条代表平均强度；误差条表示标准差。

Chromosome spreads using

使用染色体铺展

P. maniculatus

鹿鼠属

meiosis I oocytes expressing dCas9-EGFP and meiosis II oocytes expressing dCas9-mCherry together with gRNA targeting PMsat were stained with HEC1 and REC8;

表达dCas9-EGFP的减数分裂I期卵母细胞和表达dCas9-mCherry及靶向PMsat的gRNA的减数分裂II期卵母细胞一起用HEC1和REC8染色；

= 14 and 9 cells from three independent experiments were analyzed for meiosis I and II, respectively. Additional examples of REC8 staining in Supplementary Fig.

= 14，且分别从三个独立实验中分析了减数分裂I和II的9个细胞。REC8染色的更多示例见补充图。

。

P. maniculatus

鹿鼠属

meiosis I oocytes microinjected with mCherry-Trim21 mRNA together with either control IgG antibody or anti-REC8 antibody were matured to meiosis II and fixed and stained for MCAK (a PMsat marker, see Fig.

微注射了mCherry-Trim21 mRNA并与对照IgG抗体或抗REC8抗体一起处理的减数分裂I卵母细胞被培养至减数分裂II，并固定和染色以标记MCAK（一种PMsat标记，见图）。

) and HEC1. Chromosomes with a single kinetochore (HEC1) were scored as single chromatids, and the proportion of chromosomes exhibiting sister chromatid separation was quantified; each dot represents an individual experiment;

）和HEC1。具有单个动粒（HEC1）的染色体被计为单个染色单体，并量化了表现出姐妹染色单体分离的染色体比例；每个点代表一个单独的实验；

= 23 and 15 cells from three independent experiments for the IgG and REC8 antibody, respectively; red line, mean. The images are maximum projections showing all the chromosomes (left) and optical sections to show individual chromosomes (right); asterisks denote the chromosomal location of internal PMsat (orange) and telomeric PMsat (yellow) on dual PMsat chromosomes, scale bars, 5 µm.

分别来自三次独立实验的IgG和REC8抗体的23和15个细胞；红线表示平均值。图像为最大投影，显示所有染色体（左）和光学切片以显示单个染色体（右）；星号标记了双PMsat染色体上内部PMsat（橙色）和端粒PMsat（黄色）的染色体位置，比例尺为5微米。

Source data are provided as a Source Data file..

源数据作为源数据文件提供。

Full size image

全尺寸图像

Homologous chromosomes recombine and become connected by chiasmata in meiosis I. If the recombination occurred between two PMsat blocks, the cohesion at telomeric PMsat could be deleterious to the cell by preventing the separation of homologous chromosomes in anaphase I (Supplementary Fig.

同源染色体在减数分裂I中重组并通过交叉点连接。如果重组发生在两个PMsat区块之间，端粒处的PMsat粘连可能对细胞有害，因为它会阻止同源染色体在后期I中分离（补充图）。

). We did not find any oocytes with such recombination pattern in both

）。我们没有在两者中发现任何具有这种重组模式的卵母细胞

Peromyscus maniculatus

北美鹿鼠

and

和

Peromyscus polionotus

北美鹿鼠

, implying a mechanism to prevent recombinations between two PMsat blocks. (Peri)centromeric regions usually have lower recombination rates

，暗示了一种防止两个PMsat块之间重新组合的机制。（近）着丝粒区域通常具有较低的重组率。

，

, and therefore, a similar mechanism could be at play between two PMsat blocks to avoid meiotic failures.

，因此，两个PMsat块之间可能也存在类似的机制以避免减数分裂失败。

The cohesin complex mediates chromosome cohesion in mitosis and meiosis

黏连蛋白复合体在有丝分裂和减数分裂中介导染色体的黏连。

，

. The unconventional cohesion pattern observed in

. 观察到的非常规凝聚力模式

Peromyscus

鹿鼠属

oocytes raised a possibility that telomeric PMsat facilitates cohesin-mediated chromosome cohesion specifically in meiosis. To test this possibility, we first examined the localization of meiosis-specific cohesin subunit, REC8, in oocytes. In multiple organisms, cohesin localizes along the chromosome axis in meiosis I, followed by the Separase-mediated cleavage in anaphase I except for pericentromeric cohesin.

卵母细胞提出了一种可能性，即端粒PMsat在减数分裂中促进黏连蛋白介导的染色体黏连。为了验证这种可能性，我们首先检查了减数分裂特异性黏连蛋白亚基REC8在卵母细胞中的定位。在多种生物中，黏连蛋白在减数分裂I期沿染色体轴定位，随后在I期后期被分离酶介导切割，但着丝粒周围的黏连蛋白除外。

Analogous to protecting cohesin from the prophase pathway in mitosis, the SGO-PP2A complex protects pericentromeric cohesin from Separase at anaphase I.

类似于在有丝分裂中保护黏连蛋白免受前中期途径的影响，SGO-PP2A复合物在减数第一次分裂后期保护着丝粒周围的黏连蛋白免受分离酶的作用。

，

. The remaining cohesin at the pericentromere allows the bi-orientation of sister chromatids in meiosis II much like in mitosis. Consistent with other organisms, REC8 cohesin localized on the chromosome axis in meiosis I and at the pericentromere in meiosis II (Fig.

。在减数分裂II中，着丝粒周围的剩余粘连蛋白允许姐妹染色单体的双向定位，类似于有丝分裂。与其他生物一致，REC8粘连蛋白在减数分裂I中定位于染色体轴上，在减数分裂II中定位于着丝粒周围（图。

and Supplementary Fig.

和补充图。

), suggesting that general principles for meiotic cohesin regulations are conserved in

），这表明减数分裂凝聚素调控的普遍原则在

Peromyscus

鹿鼠属

mice. When we focused on dual PMsat chromosomes in meiosis II, we found that cohesin remains localized at telomeric PMsat in addition to the pericentromere (Fig.

小鼠。当我们关注减数分裂II中的双重PMsat染色体时，我们发现黏连蛋白除了在着丝粒周围外，仍然定位于端粒PMsat（图。

, meiosis II dual, and Supplementary Fig.

，减数分裂II双重，及补充图。

), consistent with our hypothesis. To directly test if cohesin mediates sister-chromatid cohesion at telomeric PMsat, we acutely degraded REC8 by the Trim-Away method

），与我们的假设一致。为了直接测试粘连蛋白是否在端粒PMsat处介导姐妹染色单体的粘连，我们通过Trim-Away方法急性降解了REC8。

，

. We found that both standard and dual PMsat chromosomes fell apart into single chromatids as evidenced by a single kinetochore on the chromosome in contrast to two kinetochores for sister-chromatid pairs in the control condition (Fig.

我们发现，标准和双PMsat染色体都分解为单个染色单体，这由染色体上的单个动粒证明，而对照条件下姐妹染色单体对则有两个动粒（图。

and Supplementary Fig.

和补充图。

). These data suggest that cohesion at telomeric PMsat is mediated by the cohesin complex. Dual PMsat chromosomes that were not completely separated by REC8 Trim-Away were predominantly connected at telomeric PMsat (Supplementary Fig.

这些数据表明，端粒PMsat的粘连是由粘连蛋白复合体介导的。那些未被REC8 Trim-Away完全分离的双PMsat染色体主要在端粒PMsat处连接（补充图）。

5天

), supporting the idea that telomeric PMsat is the major cohesion site.

），支持端粒PMsat是主要的凝聚位点这一观点。

PP2A-mediated cohesin protection at telomeric PMsat

PP2A介导的端粒PMsat处的黏连蛋白保护

Cohesin needs to be protected by PP2A activity to maintain its localization at the metaphase I—anaphase I transition

在第一次减数分裂中期到后期的过渡中，黏连蛋白需要通过PP2A活性来保护其定位。

，

. We hypothesized that PP2A enriches at telomeric PMsat, in addition to its canonical localization at the pericentromere, to protect cohesin at telomeric PMsat. Indeed, we found that PP2A localized at telomeric PMsat in meiosis I oocytes (Fig.

我们假设PP2A除了在着丝粒周围的典型定位外，还会在端粒PMsat处富集，以保护端粒PMsat处的黏连蛋白。确实，我们发现在减数分裂I的卵母细胞中，PP2A定位于端粒PMsat（图。

). PP2A levels were slightly but significantly higher at telomeric PMsat compared to internal PMsat, consistent with the observation that telomeric PMsat serves as the major cohesion site of dual PMsat chromosomes in the following meiosis II division. If telomeric PMsat is the major cohesion site enriching a higher PP2A activity, this locus should be more tolerant to the partial inhibition of the PP2A activity.

）。与内部PMsat相比，端粒PMsat处的PP2A水平略高但显著，这与端粒PMsat在随后的减数分裂II中作为双PMsat染色体主要黏连位点的观察结果一致。如果端粒PMsat是富集较高PP2A活性的主要黏连位点，那么该位点应该对PP2A活性的部分抑制更具耐受性。

To test this idea, we treated oocytes with a lower concentration of a PP2A inhibitor, Okadaic acid (OA).

为了测试这个想法，我们用较低浓度的PP2A抑制剂——冈田酸（OA）处理了卵母细胞。

(Fig.

（图。

). Upon partial PP2A inhibition, we observed a substantial increase in the number of dual PMsat sister chromatids only connected at telomeric PMsat (Fig.

). 在部分抑制PP2A后，我们观察到仅在端粒PMsat处相连的双重PMsat姐妹染色单体数量显著增加（图。

, top graph). We also noticed that dual PMsat chromosomes are more resistant to the PP2A inhibition compared to standard chromosomes (Fig.

，上图）。我们还注意到，与标准染色体相比，双PMsat染色体对PP2A抑制更具抵抗力（图。

, bottom graph). This result implies that carrying an additional block of centromeric satellite could be beneficial for the chromosome to ensure sister-chromatid cohesion during meiosis if cells could prevent the deleterious recombination pattern between two centromeric satellite blocks (Supplementary Fig. .

，下图）。该结果暗示，如果细胞能够防止两个着丝粒卫星区块之间有害的重组模式，那么携带额外一块着丝粒卫星可能对染色体确保减数分裂期间的姐妹染色单体凝聚力是有益的（补充图）。

, see Discussion).

，见讨论）。

Fig. 3: PP2A-mediated cohesin protection at telomeric PMsat.

图3：PP2A介导的端粒PMsat处的黏连蛋白保护。

Chromosome spreads of

染色体分布

P. maniculatus

鹿鼠属

meiosis I oocytes expressing dCas9-mCherry with gRNA targeting PMsat were stained for ACA (kinetochore) and PP2A. Signal intensities of PP2A at PMsat were quantified; each dot represents one chromosome;

表达dCas9-mCherry的减数分裂I期卵母细胞，其gRNA靶向PMsat，用ACA（动粒）和PP2A染色。量化了PMsat上PP2A的信号强度；每个点代表一条染色体；

= 152 chromosomes from three independent experiments; unpaired two-tailed Mann-Whitney test was used to analyze statistical significance; red line, median.

= 152 条染色体来自三个独立实验；使用未配对双尾 Mann-Whitney 检验分析统计显著性；红线表示中位数。

P. maniculatus

鹿鼠属

meiosis I oocytes expressing dCas9-EGFP with gRNA targeting PMsat were treated with 10 nM Okadaic acid (OA), matured to meiosis II, and fixed and stained for HEC1. The proportion of each chromosome configuration of dual PMsat chromosomes (top graph) and sister-chromatid separation (bottom graph; DAPI and HEC1 signals were used to determine if the chromosome is a single chromatid or sister chromatids) were quantified; each dot represents an individual experiment; .

表达dCas9-EGFP且gRNA靶向PMsat的减数分裂I期卵母细胞经10 nM冈田酸（OA）处理后，成熟至减数分裂II期，并固定和染色HEC1。对双PMsat染色体的每种染色体构型比例（上图）以及姐妹染色单体分离情况（下图；使用DAPI和HEC1信号判断染色体是单染色单体还是姐妹染色单体）进行量化；每个点代表一个独立实验。

= 26 and 33 oocytes from four independent experiments for control and the OA-treated group, respectively; unpaired two-sided t-test was used to analyze statistical significance; red line, mean. Exact

分别从四个独立实验中获取了26和33个卵母细胞作为对照组和OA处理组；使用未配对的双侧t检验分析统计显著性；红线表示平均值。精确值如下。

values are in the graphs except for

图表中除以下值之外

****P

< 0.0001; the images are maximum projections showing all the chromosomes (left) and optical sections to show individual chromosomes (right); asterisks denote the chromosomal location of internal PMsat (orange) and telomeric PMsat (yellow) on dual PMsat chromosomes; scale bars, 5 µm. Source data are provided as a Source Data file..

小于0.0001；图像为最大投影，显示所有染色体（左）和光学切片以显示单个染色体（右）；星号表示双PMsat染色体上内部PMsat（橙色）和端粒PMsat（黄色）的染色体位置；比例尺，5微米。源数据作为源数据文件提供。

Full size image

全尺寸图像

Telomeric PMsat assembles a pericentromere-like structure

端粒PMsat组装成类似着丝粒的结构

Given that cohesin and PP2A localized at telomeric PMsat, we wondered if telomeric PMsat also enriches other pericentromeric factors, assembling a pericentromere-like structure that is decoupled from the kinetochore. To test this possibility, we examined the localization of MCAK (mitotic centromere associated kinesin), which is a member of the kinesin-13 family, and the Chromosomal Passenger Complex (CPC) composed of Survivin, Borealin, INCENP, and Aurora B/C kinase.

鉴于黏连蛋白和PP2A定位于端粒的PMsat，我们想知道端粒的PMsat是否也会富集其他近着丝粒因子，从而组装成一种与动粒分离的类似近着丝粒结构。为了验证这种可能性，我们检测了MCAK（有丝分裂着丝粒相关驱动蛋白）的定位，它是驱动蛋白-13家族的成员，同时还检测了由Survivin、Borealin、INCENP和Aurora B/C激酶组成的染色体乘客复合体（CPC）。

，

四十三

，

. Survivin, phosphorylated Aurora (pAurora, labeling active CPC), and MCAK were highly enriched at telomeric PMsat in meiosis I and II oocytes in addition to their characteristic localization next to the kinetochore (Fig.

在减数分裂I和II的卵母细胞中，Survivin、磷酸化的Aurora（pAurora，标记活跃的CPC）和MCAK除了在动粒旁有特征性定位外，在端粒PMsat处也高度富集（图。

4a–c

and Supplementary Fig.

和补充图。

6a–c

). Similarly, pericentromeric factors localized at telomeric PMsat in

）。同样，位于端粒PMsat的近中心因子

Peromyscus polionotus

北美鹿鼠

oocytes (Supplementary Fig.

卵母细胞（补充图）。

7a–c

). In contrast,

). 相比之下，

Peromyscus californicus

加州白足鼠

, which does not carry dual PMsat chromosomes (Fig.

，其不携带双重PMsat染色体（图。

), showed conventional features with the kinetochore and pericentromeric factors always juxtaposed on the chromosome without the formation of additional pericentromere-like structure (Supplementary Fig.

），显示了与着丝粒和近着丝粒因子始终并列在染色体上的传统特征，没有形成额外的类似近着丝粒结构（补充图）。

7天

). Altogether, these results suggest that the extra centromeric satellite block without kinetochore proteins can recruit pericentromeric factors to establish a pericentromere-like structure, implying a genetic contribution of centromeric satellites to assemble the pericentromere.

这些结果表明，额外的着丝粒卫星区块即使没有动粒蛋白，也可以募集周围着丝粒因子来建立类似周围着丝粒的结构，这暗示着丝粒卫星对组装周围着丝粒具有遗传贡献。

Fig. 4: Telomeric PMsat assembles an ectopic pericentromere-like structure decoupled from the kinetochore.

图4：端粒PMsat组装了一个与着丝粒分离的异位近着丝粒样结构。

–

P. maniculatus

meiosis I oocytes expressing dCas9-EGFP with gRNA targeting PMsat were fixed and stained for HEC1 as well as Survivin (

表达 dCas9-EGFP 且 gRNA 靶向 PMsat 的减数分裂 I 卵母细胞被固定并染色以检测 HEC1 和 Survivin。

), phosphorylated Aurora kinase (

），磷酸化的Aurora激酶（

), and MCAK (

），以及 MCAK （

). Signal intensities of Survivin, pAurora, and MCAK at PMsat were quantified; each dot represents one chromosome;

). 对PMsat上Survivin、pAurora和MCAK的信号强度进行了量化；每个点代表一条染色体；

= 32, 210, and 167 chromosomes from three independent experiments were analyzed for Survivin, pAurora, and MCAK, respectively; unpaired two-tailed Mann-Whitney test was used to analyze statistical significance; exact

分别分析了来自三个独立实验的Survivin、pAurora和MCAK的32、210和167条染色体；使用未配对双尾Mann-Whitney检验分析统计显著性；精确值。

values are in the graphs except for

图形中除以下值外

****P

< 0.0001; red line, median. The images are maximum projections showing all the chromosomes (left) and optical sections to show individual chromosomes (right); asterisks denote the chromosomal location of internal PMsat (orange) and telomeric PMsat (yellow) on dual PMsat chromosomes; scale bars, 5 µm.

小于0.0001；红线，中位数。图像为最大投影，显示所有染色体（左）和光学切片以显示单个染色体（右）；星号表示双PMsat染色体上内部PMsat（橙色）和端粒PMsat（黄色）的染色体位置；比例尺，5微米。

Source data are provided as a Source Data file..

源数据作为源数据文件提供。

Full size image

全尺寸图像

H2A-pT121 recruits pericentromeric factors to telomeric PMsat

H2A-pT121招募着丝粒周围因子到端粒PMsat

We next asked how telomeric PMsat recruits major pericentromeric factors. While multiple inter-dependencies ensure the enrichment of pericentromeric factors, it is established that two epigenetic marks, histone H3-pT3 and H2A-pT121, are critical for enriching pericentromeric factors in multiple organisms (Fig. .

接下来，我们探讨了端粒PMsat如何招募主要的近中心体因子。尽管多种相互依赖性确保了近中心体因子的富集，但已确定的是，两种表观遗传标记——组蛋白H3-pT3和H2A-pT121——在多种生物体中对近中心体因子的富集至关重要（图）。

)

，

. Therefore, we tested if these pathways contribute to rewiring the landscape of chromosome cohesion in meiosis. First, we tested the Haspin kinase-mediated H3-pT3 pathway, which recruits CPC through the interaction with the Survivin subunit

因此，我们测试了这些通路是否有助于在减数分裂中重新构建染色体粘连的景观。首先，我们测试了Haspin激酶介导的H3-pT3通路，该通路通过与Survivin亚基的相互作用招募CPC。

. We found that inhibiting Haspin by a chemical inhibitor, 5-iodotubucidin (5-Itu), abolished H3-pT3 signals on the chromosome (Fig.

我们发现，通过化学抑制剂5-碘代秋水仙素（5-Itu）抑制Haspin后，染色体上的H3-pT3信号消失（图。

and Supplementary Fig.

和补充图。

)

. Furthermore, Haspin inhibition reduced CPC levels on the chromosome arm without significantly impacting its pericentromeric localization (Supplementary Fig.

此外，Haspin 抑制降低了染色体臂上的 CPC 水平，而对其近着丝粒定位没有显著影响（补充图）。

8b, c

8b，c

), consistent with a previous study using oocytes from lab standard mice,

），与之前使用实验室标准小鼠卵母细胞的研究一致，

Mus musculus

小家鼠

. Notably, we did not see a significant reduction in CPC and MCAK levels at telomeric PMsat (Fig.

特别地，我们没有在端粒PMsat处观察到CPC和MCAK水平的显著降低（图。

and Supplementary Fig.

和补充图。

8d, e

8天，e

). Therefore, we next tested the BUB1 kinase-mediated H2A-pT121 pathway, which interacts with SGO2 to recruit MCAK, PP2A, and CPC

因此，我们接下来测试了BUB1激酶介导的H2A-pT121通路，该通路与SGO2相互作用以招募MCAK、PP2A和CPC。

，

. H2A-pT121 signals were detected along the chromosome with a slight enrichment around the pericentromere (Fig.

H2A-pT121 信号沿染色体被检测到，在近着丝粒周围有轻微的富集（图。

5天

). The chromosomal H2A-pT121 signals were significantly reduced after inhibiting BUB1 by a chemical inhibitor, BAY-1816032 (Fig.

）。使用化学抑制剂BAY-1816032抑制BUB1后，染色体H2A-pT121信号显著减少（图。

5天

)

. The BUB1 inhibition reduced CPC levels on the chromosome (Supplementary Fig.

BUB1抑制降低了染色体上的CPC水平（补充图）。

), and importantly, reduced CPC and MCAK levels at telomeric PMsat (Fig.

），重要的是，降低了端粒PMsat处的CPC和MCAK水平（图。

5e, f

), suggesting that the BUB1-H2A-pT121 pathway drives the rewiring of the major cohesion site to telomeric PMsat.

），这表明BUB1-H2A-pT121通路驱动了主要黏连位点向端粒PMsat的重新布线。

Fig. 5: The BUB1 kinase-H2A-pT121 pathway recruits pericentromeric factors to telomeric PMsat.

图5：BUB1激酶-H2A-pT121通路将着丝粒周围因子招募到端粒PMsat。

Schematic of two pathways recruiting pericentromeric factors.

招募着丝粒周围因子的两条途径示意图。

P. maniculatus

鹿鼠属

meiosis I oocytes treated with 5-Itu were fixed at meiosis II and stained for HEC1 and H3-pT3.

用5-Itu处理的减数分裂I期卵母细胞在减数分裂II期被固定，并用HEC1和H3-pT3进行染色。

= 16, 5, and 10 cells from three independent experiments for control, 0.5 µM 5-Itu, and 1µM 5-Itu, respectively.

分别来自三次独立实验的控制组、0.5 µM 5-Itu组和1 µM 5-Itu组的细胞数为16、5和10。

P. maniculatus

鹿鼠属

meiosis I oocytes expressing dCas9-EGFP with gRNA targeting PMsat were treated with 5-Itu, fixed at metaphase I, and stained for HEC1 and Survivin. Survivin levels at telomeric PMsat were quantified; each dot represents one chromosome;

表达 dCas9-EGFP 且 gRNA 靶向 PMsat 的减数分裂 I 卵母细胞经 5-Itu 处理后，在中期 I 固定，并用 HEC1 和 Survivin 染色。端粒 PMsat 处的 Survivin 水平被量化，每个点代表一条染色体；

= 26 and 23 cells from three independent experiments for control and the 5-Itu-treated group, respectively; red line, median.

分别来自三次独立实验的对照组和5-Itu处理组的细胞数为26和23个；红线表示中位数。

P. maniculatus

meiosis I oocytes treated with BAY-1816032 were fixed at metaphase I and stained for H2A-pT121 and HEC1. H2A-pT121 levels on chromosomes were quantified; each dot represents one oocyte;

用BAY-1816032处理的减数分裂I卵母细胞在中期I被固定，并用H2A-pT121和HEC1染色。对染色体上的H2A-pT121水平进行了量化；每个点代表一个卵母细胞；

= 13 and 11 oocytes from three independent experiments for control and the BAY-1816032-treated group, respectively; unpaired two-tailed Mann-Whitney test was used for statistical analysis; red line, median.

分别从三个独立实验中获取了13个和11个卵母细胞作为对照组和BAY-1816032处理组；统计分析使用了非配对双尾Mann-Whitney检验；红线表示中位数。

P. maniculatus

meiosis I oocytes expressing dCas9-mCherry with gRNA targeting PMsat were treated with BAY-1816032, fixed at metaphase I, and stained for Survivin and HEC1. Survivin levels at telomeric PMsat were quantified; each dot represents an individual chromosome;

表达 dCas9-mCherry 且 gRNA 靶向 PMsat 的减数分裂 I 卵母细胞经 BAY-1816032 处理后，固定在中期 I，并用 Survivin 和 HEC1 染色。端粒 PMsat 处的 Survivin 水平被量化；每个点代表一条染色体；

= 16 and 10 oocytes from three independent experiments for control and the BAY-1816032-treated group, respectively; unpaired two-tailed Mann-Whitney test was used for statistical analysis; red line, median.

= 16，对照组和BAY-1816032处理组分别来自三个独立实验的10个卵母细胞；统计分析采用未配对双尾Mann-Whitney检验；红线表示中位数。

P. maniculatus

meiosis I oocytes expressing dCas9-mCherry with gRNA targeting PMsat were treated with BAY-1816032, fixed at metaphase I, and stained for MCAK and HEC1. MCAK levels at PMsat were quantified; each dot represents an individual chromosome;

表达 dCas9-mCherry 且 gRNA 靶向 PMsat 的减数分裂 I 卵母细胞经 BAY-1816032 处理后，在中期 I 固定，并用 MCAK 和 HEC1 染色。PMsat 处的 MCAK 水平被量化，每个点代表单个染色体；

= 122 and 141 chromosomes from four independent experiments for control and the BAY-1816032-treated group, respectively; unpaired two-tailed Mann-Whitney test was used for statistical analysis; red line, median. Orange asterisks, internal PMsat; yellow asterisks, telomeric PMsat; exact

分别从四个独立实验中获得对照组和BAY-1816032处理组的122和141条染色体；统计分析采用未配对双尾Mann-Whitney检验；红线表示中位数。橙色星号表示内部PMsat；黄色星号表示端粒PMsat；精确值。

values are in the graphs except for

图形中除……外的值

****P

< 0.0001; scale bars, 5 µm. Source data are provided as a Source Data file.

小于0.0001；比例尺，5微米。源数据作为源数据文件提供。

Full size image

全尺寸图像

Formation of ectopic additional cohesion sites is specific to meiosis

异位额外粘连位点的形成是减数分裂特有的。

While telomeric PMsat serves as the major cohesion sites for dual PMsat chromosomes in female meiosis, sister chromatids appear mainly cohered at internal PMsat in mitosis (Fig.

虽然端粒PMsat在雌性减数分裂中作为双PMsat染色体的主要黏连位点，但在有丝分裂中，姐妹染色单体似乎主要在内部PMsat处保持黏连（图。

). To understand the mechanisms underlying the difference between mitosis and meiosis, we examined the localization pattern of pericentromeric factors in mitosis, using ovarian granulosa cells. We found that pericentromeric factors and H2A-pT121 were restricted to internal PMsat, localizing between sister kinetochores (Fig. .

为了理解有丝分裂和减数分裂之间差异的潜在机制，我们使用卵巢颗粒细胞检查了有丝分裂中着丝粒周围因子的定位模式。我们发现着丝粒周围因子和H2A-pT121局限于内部的PMsat，定位在姐妹动粒之间（图. 。

6a–c

), similar to the observations in other model organisms

)，与在其他模式生物中的观察结果相似

，

. The absence of pericentromeric factors would lead to de-protection of cohesin at telomeric PMsat, explaining why dual PMsat chromosomes are cohered at internal PMsat in mitosis (Fig.

着丝粒周围因子的缺失会导致端粒PMsat处黏连蛋白的去保护，这解释了为什么在有丝分裂中，双重PMsat染色体会在内部PMsat处保持黏连（图。

6天

). Consistent with this result, we confirmed that pericentromeric factors localize between sister kinetochores and do not form ectopic telomere-proximal pericentromere-like structures in bone marrow mitotic cells (Supplementary Fig.

与此结果一致，我们确认了着丝粒周围因子定位于姐妹动粒之间，在骨髓有丝分裂细胞中不会形成异位的靠近端粒的类着丝粒结构（补充图）。

）。

Fig. 6: The formation of ectopic additional cohesion sites is specific to meiosis.

图6：异位额外粘连位点的形成是减数分裂特有的。

–

P. maniculatus

cells (granulosa cells) arrested in mitosis by Nocodazole were fixed and stained for HEC1 together with Survivin (

被诺考达唑阻滞在有丝分裂中的细胞（颗粒细胞）被固定并用HEC1和Survivin染色（

), phosphorylated Aurora kinase (

），磷酸化的Aurora激酶（

), or H2A-pT121 (

)，或 H2A-pT121 (

). Immunostained cells were then labeled for PMsat using the Oligopaint technique.

免疫染色的细胞随后使用Oligopaint技术标记PMsat。

= 13, 21, and 11 cells from three independent experiments were analyzed. Line scans of the signal intensities of Survivin (

= 13、21和11个细胞来自三个独立实验的分析。对Survivin信号强度进行了线扫描 (

), pAurora (

)，pAurora（

), or H2A-pT121 (

)，或 H2A-pT121 (

) together with PMsat and HEC1 were performed along the chromosome. Signal intensities of Survivin (

) 与 PMsat 和 HEC1 沿着染色体进行。Survivin 的信号强度 (

), pAurora (

)，pAurora（

), and H2A-pT121 (

)，以及 H2A-pT121 (

) at PMsat were quantified; each dot represents one chromosome;

) 在 PMsat 上被量化；每个点代表一条染色体；

= 197, 147, and 128 chromosomes from three independent experiments were analyzed for Survivin, pAurora, and MCAK, respectively; unpaired two-tailed Mann-Whitney test was used to analyze statistical significance,

分别分析了来自三个独立实验的197、147和128条染色体的Survivin、pAurora和MCAK；采用非配对双尾Mann-Whitney检验分析统计学显著性，

****P

< 0.0001; red line, median. The images are maximum projections showing all the chromosomes (top) and optical sections to show individual chromosomes (bottom); asterisks denote the chromosomal location of internal PMsat (orange) and telomeric PMsat (yellow) on dual PMsat chromosomes; scale bars, 5 µm.

小于0.0001；红线，中位数。图像为最大投影，显示所有染色体（顶部）和光学切片以显示单个染色体（底部）；星号表示双PMsat染色体上内部PMsat（橙色）和端粒PMsat（黄色）的染色体位置；比例尺，5微米。

。

, Model for the centromere and pericentromere specification in mitosis and meiosis when a chromosome carries single or dual centromere satellite block. Source data are provided as a Source Data file.

，当染色体携带单个或双中心粒卫星块时，有丝分裂和减数分裂中的着丝粒和近着丝粒特异性模型。源数据作为源数据文件提供。

Full size image

全尺寸图像

Discussion

讨论

We have uncovered that centromeric satellites facilitate sister-chromatid cohesion, using

我们发现着丝粒卫星促进姐妹染色单体的粘连，通过使用

Peromyscus

鹿鼠属

mice. Because centromeres and kinetochores are usually colocalized, it has been challenging to investigate non-kinetochore roles of centromeric satellites. The unique centromere organization in

小鼠。由于着丝粒和动粒通常共定位，研究着丝粒卫星的非动粒角色一直具有挑战性。独特的着丝粒组织结构在

Peromyscus

鹿鼠属

mice allowed us to tackle this question, leading to the identification of the meiosis-specific formation of additional cohesion sites on centromeric satellites that are spatially separated from the kinetochore. The histone H2A-pT121 mark was identified as the main driver to assemble this pericentromere-like structure, recruiting pericentromeric factors such as PP2A, CPC, and MCAK.

小鼠使我们能够解决这个问题，从而鉴定出在着丝粒卫星上减数分裂特异性形成的额外粘连位点，这些位点在空间上与动粒分离。组蛋白H2A-pT121标记被确定为组装这种类似着丝粒结构的主要驱动因素，招募着丝粒周围因子如PP2A、CPC和MCAK。

While it is established that centromeric satellites are not sufficient to establish centromere identity.

虽然已确定着丝粒卫星不足以建立着丝粒身份。

, our study implies a previously unappreciated role of centromeric satellites in conferring pericentromere identity. Previous studies on neocentromeres mostly focused on the kinetochore position. Results from this study highlight the importance of revisiting chromosomes with neocentromeres to examine if the original centromeric satellite still enriches pericentromeric factors and contributes to sister-chromatid cohesion in mitosis and meiosis.

，我们的研究暗示着中心粒卫星在赋予近中心体身份方面的一个以前未被重视的角色。以往关于新中心体的研究主要集中在动粒的位置上。本研究的结果强调了重新审视具有新中心体的染色体的重要性，以检查原始的中心粒卫星是否仍然富集近中心体因子，并在有丝分裂和减数分裂中促进姐妹染色单体的黏连。

Indeed, one study has shown that CPC does not fully relocate to the neocentromere from the original centromere in human patient cell lines.

事实上，一项研究表明，在人类患者细胞系中，CPC并不会完全从原来的着丝粒重新定位到新着丝粒。

. Our study revealed a remarkable flexibility in regulating pericentromeric factors in contrast to the stable specification of centromeres and raises two fundamental questions: (1) how is the pericentromere-like structure established in a meiosis-specific manner and (2) Is there an evolutionary advantage to harbor two blocks of centromeric satellites on a single chromosome, and what is it if so?.

我们的研究揭示了调节近中心体因素的显著灵活性，相比之下，中心体的特化是稳定的，并提出了两个基本问题：(1) 类似近中心体的结构是如何以减数分裂特异性方式建立的？(2) 在单一染色体上存在两段中心体卫星序列是否具有进化优势，如果有的话，那是什么？

Our results suggest that H2A-pT121 spreads to the entire chromosome including telomeric PMsat specifically in meiosis to form the additional cohesion site. However, it remains unknown how this epigenetic mark spreads to the entire chromosome in meiosis. BUB1 kinase, which phosphorylates H2A, is restricted to the kinetochore in both mitosis and meiosis (Supplementary Fig. .

我们的结果表明，H2A-pT121在减数分裂中特异性地扩展到包括端粒PMsat在内的整个染色体，以形成额外的黏连位点。然而，这种表观遗传标记如何在减数分裂中扩展到整个染色体尚不清楚。磷酸化H2A的BUB1激酶在有丝分裂和减数分裂中都局限于着丝粒（补充图）。

) and therefore does not explain the H2A-pT121 spreading. It has been shown that cytoplasmic BUB1 can recruit SGO (the PP2A partner) to chromatin

）因此无法解释H2A-pT121的扩散。已有研究表明，细胞质中的BUB1可以将SGO（PP2A的伴侣）招募到染色质。

. Therefore,

因此，

Peromyscus

鹿鼠属

oocytes might have overall higher BUB1 activity, which allows BUB1 to act both locally and globally. Phosphorylation levels depend on the balance between the kinase and phosphatase activities. Thus, another possibility is that the activity of the phosphatase that dephosphorylates H2A is relatively weaker in meiosis compared to mitosis.

卵母细胞可能整体上具有更高的BUB1活性，这使得BUB1能够局部和全局地发挥作用。磷酸化水平取决于激酶和磷酸酶活性之间的平衡。因此，另一种可能性是，在减数分裂中，相对于有丝分裂，使H2A去磷酸化的磷酸酶的活性相对较弱。

The H2A-pT121 spreading has also been observed in .

H2A-pT121 的扩散也已被观察到。

Mus musculus

小家鼠

oocytes

卵母细胞

. It would be interesting to explore the biological significance of this drastic change in the epigenetic pattern. In addition, H2A-pT121 is required but not sufficient to explain why pericentromeric factors are restricted to telomeric PMsat. It is likely that there are other factors (e.g., heterochromatin marks) enriched at telomeric PMsat that bridge telomeric PMsat and the formation of the pericentromere-like structure.

探索这种表观遗传模式的剧烈变化的生物学意义将会很有趣。此外，H2A-pT121是必需的，但不足以解释为什么着丝粒周围因子局限于端粒附近的PMsat。很可能存在其他在端粒附近PMsat富集的因素（例如，异染色质标记），这些因素将端粒附近的PMsat与着丝粒样结构的形成联系起来。

Future studies would reveal other requirements that are critical to rewire the cohesion landscape in meiosis..

未来的研究将揭示其他对减数分裂中重排凝聚景观至关重要的要求。

It is interesting to speculate why telomeric PMsat serves as a cohesion site in a meiosis-specific manner. In contrast to mitosis, meiosis undergoes the characteristic two step removal of cohesin from the chromosome. Failure to properly protect cohesin at the pericentromere in anaphase I would result in producing aneuploid gametes and reducing fertility.

有趣的是，端粒PMsat为何在减数分裂中特异性地充当黏连位点。与有丝分裂不同，减数分裂经历了从染色体上分两步去除黏连蛋白的特征过程。在第一次减数分裂后期，如果未能正确保护着丝粒周围的黏连蛋白，将导致产生非整倍体配子并降低生育能力。

Furthermore, current and previous studies have shown that sister-chromatid cohesion is in general weaker in meiosis II compared to mitosis.

此外，当前和以前的研究表明，与有丝分裂相比，姐妹染色单体在减数分裂II中的粘连通常较弱。

，

(Supplementary Fig.

（补充图。

). Therefore, one reason to have an additional cohesion site at telomeric PMsat is to ensure sister-chromatid cohesion until meiosis II. Aging reduces cohesin levels on meiotic chromosomes in oocytes, leading to their precocious separation especially for smaller chromosomes

因此，在端粒附近的PMsat上拥有一个额外的凝聚位点的原因之一是确保姐妹染色单体的粘连直到减数分裂II。衰老会降低卵母细胞中减数分裂染色体上的黏连蛋白水平，导致它们过早分离，尤其是较小的染色体。

，

. We noticed that smaller chromosomes tend to have two centromeric satellite blocks in

我们注意到较小的染色体往往在两条染色体块中有两个着丝粒卫星区块。

Peromyscus

鹿鼠属

mice (Fig.

小鼠（图。

). Therefore, it is an intriguing possibility that the extra centromeric satellite block serves as a backup mechanism for smaller chromosomes to ensure their sister-chromatid cohesion especially in aged mice. Reinforced cohesion of smaller chromosomes might help to extend the reproductive lifespan of female mice, as it would prevent deleterious mis-segregation of chromosomes during the meiotic divisions.

因此，一个有趣的可能性是额外的着丝粒卫星区块作为较小染色体的备份机制，以确保它们的姐妹染色单体在老年小鼠中保持粘连。加强较小染色体的粘连可能有助于延长雌性小鼠的生殖寿命，因为这将防止在减数分裂过程中染色体的有害错配。

It would be an exciting future avenue to test this idea by removing telomeric PMsat on dual PMsat chromosomes and assess its impact on reproductive lifespan..

通过去除双PMsat染色体上的端粒PMsat来测试这一想法，并评估其对生殖寿命的影响，这将是一个令人兴奋的未来研究方向。

Expanded centromeric satellites bias their transmission in animals and plants

扩展的着丝粒卫星序列在动植物中偏向其传递

，

. Thus, another possibility is that the additional centromeric satellite block increases the selfishness of the chromosome. MCAK and CPC are the major microtubule-destabilizing activity at the pericentromere that have essential roles to cancel erroneous kinetochore-microtubule attachments

因此，另一种可能性是额外的着丝粒卫星区块增加了染色体的自私性。MCAK 和 CPC 是在着丝粒周围区域具有主要微管去稳定活性的因子，它们在消除错误的动粒-微管连接中起着关键作用。

，

. This same microtubule-destabilizing activity confers selfishness to mouse centromeres, leading to biased segregation of selfish centromeres

这种相同的微管去稳定活性使小鼠着丝粒具有自私性，导致自私着丝粒的偏向性分离。

，

六十二

. Therefore, dual PMsat chromosomes might have been fixed in the population because of their ability to bias their transmission rather than being beneficial to the host.

因此，双重PMsat染色体可能因其能够偏向自身传递而非对宿主有利而在群体中被固定下来。

Altogether, this work provides a conceptual framework to investigate evolutionary forces that shape centromere organization and create karyotypic diversity.

总的来说，这项工作提供了一个概念框架，用于研究塑造着丝粒组织和创造核型多样性的进化力量。

Limitations of the study: an ideal experiment to show the significance of REC8 cohesin at telomeric PMsat would be to specifically degrade REC8 at telomeric PMsat by the REC8-TEV protease system previously established in lab standard mice

研究的局限性：一个理想的实验来展示REC8黏连蛋白在端粒PMsat处的重要性，是通过实验室标准小鼠中先前建立的REC8-TEV蛋白酶系统，特异性降解端粒PMsat处的REC8。

. However, such experiment requires generating transgenic

然而，这样的实验需要生成转基因

Peromyscus

鹿鼠属

mice, which is technically challenging at the moment

小鼠，这在技术上目前具有挑战性

. Transgenic

转基因的

Peromyscus

鹿鼠属

mice would also allow us to manipulate PMsat and robustly visualize weaker REC8 signals in meiosis II

小鼠还将使我们能够操纵PMsat，并在减数分裂II中强有力地可视化较弱的REC8信号。

(Fig.

（图。

). It remains unknown why internal PMsat has a slightly but significantly weaker cohesion compared to centromeres of standard chromosomes despite the enrichment of similar PP2A levels (Fig.

）。尽管内部PMsat中PP2A水平类似，但其凝聚力为何比标准染色体的着丝粒略弱且显著较弱仍不得而知（图。

and Supplementary Fig.

和补充图。

). We speculate that other cohesin regulators such as I2PP2A/SET, which inhibits PP2A activity

）。我们推测其他黏连蛋白调节因子，如I2PP2A/SET，它抑制PP2A的活性

, could be differentially regulated between dual PMsat and standard chromosomes.

可能在双重PMsat和标准染色体之间受到差异调控。

Methods

方法

Mouse strains

小鼠品系

Peromyscus maniculatus bairdii

贝氏鹿鼠

(BW strain),

（BW品系），

Peromyscus polionotus subgriseus

灰腹鹿鼠

(PO strain) and

（PO 应变）和

Peromyscus californicus insignis

加州鹿鼠亚种

(IS strain) mice were obtained from the

(IS菌株)小鼠获自

Peromyscus

鹿鼠属

Genetic Stock Center at the University of South Carolina (

南卡罗来纳大学遗传资源中心 (

https://sc.edu/study/colleges_schools/pharmacy/centers/peromyscus_genetic_stock_center/

). Mice were housed in an animal facility with the light/dark cycle of 12 h each and at room temperature with minimal disturbance with a range of 30-70% humidity depending on the season. Mice were euthanized with CO

）。小鼠饲养在动物设施中，昼夜周期各为12小时，室温下湿度根据季节在30-70%范围内，尽量减少干扰。小鼠用二氧化碳安乐死。

followed by cervical dislocation prior to dissection of ovaries. All animal experiments were approved by the Animal Care and Use Committee (National Institutes of Health Animal Study Proposal#: H-0327) and were consistent with the National Institutes of Health guidelines.

随后在摘除卵巢前进行颈椎脱位。所有动物实验均经动物护理和使用委员会（国立卫生研究院动物研究提案号：H-0327）批准，并符合国立卫生研究院指南。

Somatic cell isolation and culture

体细胞分离与培养

Ovarian granulosa cells and bone marrow cells were used in this study to examine mitosis. Granulosa cells were used in most experiments because of their ability to proliferate robustly after isolation.

本研究使用了卵巢颗粒细胞和骨髓细胞来观察细胞有丝分裂。由于颗粒细胞在分离后具有较强的增殖能力，因此在大多数实验中都使用了颗粒细胞。

The procedure for isolating and culturing ovarian granulosa cells has been described previously

分离和培养卵巢颗粒细胞的程序已有描述。

. Briefly, after euthanizing the mice, their ovaries were collected and rinsed three times with M2 media (Sigma-Aldrich, cat# M7167) to remove any adherent fat tissue. The ovaries were then mechanically disrupted to release oocytes and granulosa cells. Following the collection of oocytes, the remaining granulosa cells were collected into a 15 ml tube and allowed to settle at the bottom for 5–10 min.

简言之，处死小鼠后，收集其卵巢，并用M2培养基（Sigma-Aldrich，目录号# M7167）冲洗三次，以去除附着的脂肪组织。然后机械性破坏卵巢，释放卵母细胞和颗粒细胞。收集卵母细胞后，将剩余的颗粒细胞收集到15毫升试管中，静置5-10分钟使其沉降到管底。

The supernatant was discarded to remove blood cells, and the granulosa cells were then centrifuged at 500 × g for 5 min. The cells were washed extensively with DMEM high glucose GlutaMAX media (Gibco, cat# 10566-016) supplemented with 1× Antibiotic-Antimycotic (Gibco, cat# 15240062). Cells were dispersed by pipetting, washed for two additional times, and then seeded at a density of 0.6 × 10.

弃去上清液以去除血细胞，然后将颗粒细胞以500×g离心5分钟。用含1×抗生素-抗真菌剂（Gibco，货号15240062）的DMEM高糖GlutaMAX培养基（Gibco，货号10566-016）充分洗涤细胞。通过移液使细胞分散，再洗涤两次，然后以0.6×10的密度接种。

cells/ml in 6-well tissue culture-treated plates (Corning, cat# 353046) with DMEM supplemented with 10% FBS (Gibco, cat# A3160501) and 1x Antibiotic-Antimycotic. Cells were cultured in a humidified atmosphere containing 5% CO

每毫升细胞数接种于6孔组织培养处理板（Corning，货号353046），使用添加10%胎牛血清（Gibco，货号A3160501）和1倍抗生素-抗真菌剂的DMEM培养基。细胞在含5%二氧化碳的湿润环境中培养。

at 37 °C. After 24 h, the medium was replaced with fresh media of the same type to continue the primary culture for ChIP and immunostaining experiments. For immunostaining experiments, we seeded the cells on glass bottom chamber slides (Lab-Tek, cat# 155411) and enriched mitotic cells by double thymidine block and release.

在37°C下。24小时后，将培养基替换为相同类型的新鲜培养基，以继续用于ChIP和免疫染色实验的原代培养。对于免疫染色实验，我们将细胞接种在玻璃底腔室载玻片（Lab-Tek，产品编号155411）上，并通过双胸苷阻断和释放来富集有丝分裂细胞。

At the second thymidine release, 1 μM nocodazole (Sigma-Aldrich, cat# 487929-10MG-M) was added to the medium and the cells were cultured for 16 h before proceeding to standard whole-mount immunostaining or chromosome spread (see below)..

在第二次胸苷释放时，向培养基中加入1 μM的诺考达唑（Sigma-Aldrich，目录号487929-10MG-M），细胞继续培养16小时，然后进行标准的全装免疫染色或染色体铺展（见下文）。

The procedure for isolating bone marrow cells was previously described

分离骨髓细胞的程序之前已描述过。

, Briefly, bone marrow cells were collected from the femur by inserting a 26-G syringe needle into the cut end of the marrow cavity. Cells were flushed out into 3 ml of pre-warm DMEM high glucose GlutaMAX media (Gibco, cat# 10566-016) supplemented with 10% FBS (Gibco, cat# 10082147), 1 mM sodium pyruvate (Corning, cat# 25-000-CL), and 1× Antibiotic-Antimycotic solution and incubated for 2 h at 37 °C in a humidified atmosphere of 5% CO.

简而言之，通过将26-G注射器针头插入骨髓腔的切端，从股骨中收集骨髓细胞。细胞被冲入3毫升预热的DMEM高糖GlutaMAX培养基（Gibco，货号10566-016），其中添加了10%胎牛血清（FBS，Gibco，货号10082147）、1 mM丙酮酸钠（Corning，货号25-000-CL）和1×抗生素-抗真菌溶液，并在37°C、5% CO₂的湿润环境中孵育2小时。

in air. The cells were pelleted twice at 500 x g for 5 min and resuspended in 1× PBS before proceeding chromosome spread (see below).

在空气中。细胞以500 x g离心两次，每次5分钟，并在进行染色体铺展前重悬于1× PBS中（见下文）。

Chromatin extraction and ChIP-seq experiment

染色质提取和ChIP-seq实验

Granulosa cells were harvested, resuspended in 1x PBS, counted and fixed in 1% formaldehyde for 10 min at room temperature with gentle mixing. Fixation was quenched with 0.4 M glycine for 5 min at room temperature with gentle mixing. Cells were washed twice with cold 1× PBS and cell pellet was frozen on dry ice and stored at -80 °C..

颗粒细胞被收集后，重悬于1倍PBS中，计数，并在室温下用1%甲醛固定10分钟，同时轻轻混匀。固定反应使用0.4 M甘氨酸在室温下淬灭5分钟，同时轻轻混匀。细胞用冷的1倍PBS洗涤两次，细胞沉淀在干冰上冷冻并储存于-80°C。

Cells were lysed in cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1× EDTA-free protease inhibitor cocktail (Roche, cat# 5056489001)) for 10 min on ice and homogenized using type-B dounce homogenizer. Released nuclei were pelleted and lysed in nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1× EDTA-free protease inhibitor cocktail) to release chromatin.

细胞在细胞裂解缓冲液（5 mM PIPES pH 8.0，85 mM KCl，0.5% NP-40，1×无EDTA蛋白酶抑制剂混合物（Roche，货号# 5056489001））中冰上裂解10分钟，并使用B型Dounce匀浆器进行匀浆。释放的细胞核被沉淀并在核裂解缓冲液（50 mM Tris-HCl pH 8.0，150 mM NaCl，2 mM EDTA pH 8.0，1% NP-40，0.5%脱氧胆酸钠，0.1% SDS，1×无EDTA蛋白酶抑制剂混合物）中裂解以释放染色质。

Chromatin was sonicated with a Bioruptor® 300 (Diogenode), nuclear debris were then pelleted at 14000 rpm for 15 min at 4 °C, and supernatant was used for chromatin immunoprecipitation. Sonicated chromatin from 20 million cells was used for each ChIP. Dynabeads™ Protein A magnetic beads (Invitrogen, cat# 10002D) were incubated with either custom guinea pig anti-CENP-A (see below) or guinea pig IgG (SinoBiological, cat# CR4) antibodies and washed in 0.5% BSA in 1× PBS.

使用Bioruptor® 300（Diogenode）对染色质进行超声处理，然后在4°C下以14000转/分钟离心15分钟沉淀核碎片，上清液用于染色质免疫沉淀。每个ChIP使用来自2000万个细胞的超声处理染色质。Dynabeads™ Protein A磁珠（Invitrogen，货号10002D）与定制的豚鼠抗CENP-A抗体（见下文）或豚鼠IgG抗体（SinoBiological，货号CR4）孵育，并用含0.5% BSA的1× PBS洗涤。

ChIP was performed overnight on rotation at 4 °C. To generate the CENP-A antibody, mixture of two synthetic antigen peptides, MGPRRKPRTPTRRPASC and CRPSSPTPEPSRRSSHL from .

ChIP 在 4°C 下旋转过夜进行。为了生成 CENP-A 抗体，混合了两种合成抗原肽，MGPRRKPRTPTRRPASC 和 CRPSSPTPEPSRRSSHL。

Peromyscus maniculatus

鹿鼠

CENP-A N-terminal tail, were conjugated with KLH for immunization into three guinea pigs (LabCorp).

CENP-A N末端尾部与KLH结合，用于免疫三只豚鼠（LabCorp）。

Beads were then washed once with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), twice with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), twice with LiCl wash buffer (250 mM LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1 mM EDTA pH 8.0, 10 mM Tris-HCl pH 8.0) and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0).

然后用低盐洗涤缓冲液（0.1% SDS、1% Triton X-100、2 mM EDTA pH 8.0、20 mM Tris-HCl pH 8.0、150 mM NaCl）洗涤珠子一次，用高盐洗涤缓冲液（0.1% SDS、1% Triton X-100、2 mM EDTA pH 8.0、20 mM Tris-HCl pH 8.0、500 mM NaCl）洗涤两次，用LiCl洗涤缓冲液（250 mM LiCl、1% NP-40、1% 脱氧胆酸钠、1 mM EDTA pH 8.0、10 mM Tris-HCl pH 8.0）洗涤两次，最后用TE缓冲液（10 mM Tris-HCl pH 8.0、1 mM EDTA pH 8.0）洗涤两次。

Beads were incubated overnight at 65 °C in elution buffer (10 mM Tris-HCl pH 8.0, 0.3 M NaCl, 5 mM EDTA pH 8.0, 0.5% SDS) with 0.1 µg/µl RNase A (Thermo Scientific, cat# EN0531). Eluates were transferred to fresh tubes and incubated for 2 h at 55°C with 0.3 µg/µl proteinase K (Roche, cat# 3115852001).

珠子在洗脱缓冲液（10 mM Tris-HCl pH 8.0，0.3 M NaCl，5 mM EDTA pH 8.0，0.5% SDS）中与0.1 µg/µl的RNase A（Thermo Scientific，货号# EN0531）一起于65°C孵育过夜。将洗脱物转移至新管中，并与0.3 µg/µl的蛋白酶K（Roche，货号# 3115852001）一起在55°C孵育2小时。

For the chromatin input sample, elution buffer was added to an aliquot of sonicated chromatin and the sample was treated similarly to ChIP samples. DNA was finally purified with the DNA Clean & Concentrator-5 kit (Zymo Research, cat# D4004)..

对于染色质输入样本，将洗脱缓冲液加入到一部分超声处理的染色质中，并且该样本的处理方式与ChIP样本类似。最后使用DNA Clean & Concentrator-5试剂盒（Zymo Research，货号D4004）纯化DNA。

DNA libraries for NGS were obtained with the ThruPLEX® DNA-Seq Kit (Takara, cat# R400676) with DNA Single Index Kit -12S Set A (Takara, cat# R400695), following manufacturer instructions. Samples were sequenced as 100 bp paired-end reads on an Illumina NovaSeq 6000 system.

使用ThruPLEX® DNA-Seq Kit（Takara，货号 R400676）和DNA Single Index Kit -12S Set A（Takara，货号 R400695）按照制造商说明获得了用于NGS的DNA文库。样本在Illumina NovaSeq 6000系统上以100 bp双端读长进行测序。

CENP-A ChIP-seq analysis

CENP-A 染色质免疫沉淀测序分析

Read quality was assessed by fastQC v0.12.1. Reads were aligned to the genome assembly HU_Pman_2.1.3 (GCF_003704035.1) of

读取质量通过fastQC v0.12.1进行评估。读取结果比对到基因组组装HU_Pman_2.1.3 (GCF_003704035.1)。

Peromyscus maniculatus bairdii

贝氏鹿鼠

using the Burrows-Wheeler Alignment (BWA) tool v0.7.17 (bwa aln and bwa sampe commands, default settings)

使用Burrows-Wheeler比对工具（BWA）v0.7.17版（bwa aln 和 bwa sampe 命令，默认设置）

. Sam files were then converted into bam files with SAMtools v1.19

然后使用 SAMtools v1.19 将 Sam 文件转换为 Bam 文件

, while removing eventually unmapped and duplicated reads, and retaining only primary alignments (samtools view -F 0 × 4, 0 × 400, 0 × 100, 0 × 800 -b -h file.sam > file.bam). Bam files were sorted and indexed with SAMtools and converted to bigwig normalized to 1x genome coverage (RPGC normalization) for each sample with deepTools v3.5.4a.

，在去除最终未映射和重复的读取的同时，仅保留主要比对结果（samtools view -F 0×4, 0×400, 0×100, 0×800 -b -h file.sam > file.bam）。使用SAMtools对Bam文件进行排序和索引，并通过deepTools v3.5.4a将每个样本转换为归一化到1倍基因组覆盖度（RPGC归一化）的bigwig格式。

(bamCoverage --bam file.bam -o file.bw -of bigwig --binSize 10 --effectiveGenomeSize 2385634842 --normalizeUsing RPGC --extendReads 200). The effective genome size was calculated using the unique-kmers.py command of the tool khmer v2.1.1 (with -k 200)

(bamCoverage --bam file.bam -o file.bw -of bigwig --binSize 10 --effectiveGenomeSize 2385634842 --normalizeUsing RPGC --extendReads 200)。有效基因组大小使用 khmer v2.1.1 工具的 unique-kmers.py 命令计算（使用 -k 200 参数）。

，

. ChIP bigwigs were further normalized by the input using deepTools (bigwigCompare -b1 ChIP.bw -b2 input.bw -o CENPA_input_ratio.bw -of bigwig --operation ratio –skipZeroOverZero --binSize 10). Peaks were called using MACS2 v2.2.7.1

ChIP bigwigs 使用 deepTools 进一步通过 input 进行标准化（bigwigCompare -b1 ChIP.bw -b2 input.bw -o CENPA_input_ratio.bw -of bigwig --operation ratio –skipZeroOverZero --binSize 10）。使用 MACS2 v2.2.7.1 调用峰值。

(macs2 callpeak -t ChIP.bam -c input.bam -f BAMPE -g 2385634842). Heatmaps and enrichment profiles were plotted using deepTools.

(macs2 callpeak -t ChIP.bam -c input.bam -f BAMPE -g 2385634842)。热图和富集曲线使用 deepTools 绘制。

Sequences underlying CENP-A peaks were extracted with getfasta command from BEDTools v2.31.1

使用BEDTools v2.31.1中的getfasta命令提取了CENP-A峰下的序列。

. De novo motif finding was performed with Multiple Em for Motif Elicitation (MEME) tool from MEME Suite v5.5.5

使用MEME Suite v5.5.5中的Multiple Em for Motif Elicitation (MEME)工具进行从头模体查找。

(with options -mod anr -nmotifs 30 -minw 20 -maxw 50 -objfun classic -revcomp -markov_order 0).

（使用选项 -mod anr -nmotifs 30 -minw 20 -maxw 50 -objfun classic -revcomp -markov_order 0）。

To perform enrichment analysis of CENP-A at genomic regions presenting PMsat sequences, a blastn search was performed for the PMsat consensus in the HU_Pman_2.1.3 reference genome assembly. Alignment regions that overlapped or that were at most 10 bp apart were merged using BEDTools merge command. Local Z-score analysis and permutation test (.

为了在含有PMsat序列的基因组区域对CENP-A进行富集分析，针对HU_Pman_2.1.3参考基因组组装中的PMsat共识序列进行了blastn搜索。使用BEDTools merge命令将重叠或间隔至多10 bp的比对区域合并。局部Z分数分析和置换检验（。

= 1000) to assess the association between CENP-A enriched regions and PMsat regions were performed with regioneR v4.3.1

= 1000) 用于评估 CENP-A 富集区域与 PMsat 区域之间的关联，使用了 regioneR v4.3.1 进行分析。

。

Oocyte collection and maturation

卵母细胞的收集和成熟

Oocyte collection was performed as described previously

卵母细胞收集按照先前描述的方法进行。

. Oocytes were handled using a mouth-operated plastic pipette equipped with pipette tips of 75, 100, or 125 µm diameter (Cooper Surgical, Inc., cat# MXL3-75, MXL3-100, and MXL3-125). For in vitro oocyte culture, nuclear envelope (NE)-intact oocytes from female

卵母细胞使用配备有75、100或125微米直径吸头的口操塑料移液器进行操作（Cooper Surgical公司，产品编号MXL3-75、MXL3-100和MXL3-125）。对于体外卵母细胞培养，使用来自雌性小鼠的核膜（NE）完整的卵母细胞。

Peromyscus

鹿鼠属

mice were collected in M2 media (Sigma-Aldrich, cat# M7167) supplemented with 5 µM milrinone (Sigma, cat# 475840) to prevent meiotic resumption. The oocytes were washed several times in M16 media (Millipore, cat# M7292) to wash out milrinone and transferred to M16 media covered with paraffin oil (Nacalai, cat# NC1506764) to be incubated at 37 °C in a humidified atmosphere with 5% CO.

小鼠在添加了5 µM米力农（Sigma，目录号475840）的M2培养基（Sigma-Aldrich，目录号M7167）中收集，以防止减数分裂恢复。卵母细胞在M16培养基（Millipore，目录号M7292）中多次洗涤以去除米力农，然后转移到用石蜡油（Nacalai，目录号NC1506764）覆盖的M16培养基中，在37°C、5% CO₂的湿润环境中孵育。

. Only oocytes that underwent nuclear envelope breakdown (NEBD) within 90 min post-release were used for the experiments. For analyses in meiosis I, oocytes were matured for 3-7 h post-release, and meiosis II analyses were performed at least 12 h post-release. Chemical inhibitors were added to the media upon NEBD; BUB1 inhibitor, BAY-1816032 (MedChem Express, cat# HY-103020), at 10 µM; Haspin inhibitor, 5-iodotubercidin (5-Itu) (Cayman Chemical, cat# 10010375), at 0.5 or 1 µM; PP2A inhibitor, Okadaic acid (Sigma-Aldrich, cat# O9381-25UG), at 10 nM..

只有在释放后90分钟内发生核膜破裂（NEBD）的卵母细胞被用于实验。对于减数分裂I的分析，卵母细胞在释放后成熟3-7小时，而减数分裂II的分析则在释放后至少12小时进行。化学抑制剂在NEBD时添加到培养基中；BUB1抑制剂BAY-1816032（MedChem Express，目录号HY-103020），浓度为10 µM；Haspin抑制剂5-碘结核菌素（5-Itu）（Cayman Chemical，目录号10010375），浓度为0.5或1 µM；PP2A抑制剂冈田酸（Sigma-Aldrich，目录号O9381-25UG），浓度为10 nM。

Oocyte microinjection

卵母细胞显微注射

Nuclear envelope-intact oocytes were microinjected with ~5 pl of cRNAs or antibodies in M2 media containing 5 µM milrinone, using a micromanipulator TransferMan 4r and FemtoJet 4i (Eppendorf). Following the microinjection, oocytes were maintained at prophase I in M16 supplemented with 5 µM milrinone for 2-3 h to allow protein expression.

核膜完整的卵母细胞在含有5 µM米力农的M2培养基中，使用显微操作仪TransferMan 4r和FemtoJet 4i（Eppendorf）显微注射了约5 pl的cRNA或抗体。显微注射后，卵母细胞在补充有5 µM米力农的M16培养基中维持于第一次减数分裂前期2-3小时，以允许蛋白质表达。

EGFP-BUB1 (.

EGFP-BUB1 (。

Peromyscus maniculatus

北美鹿鼠

BUB1 with EGFP at the N-terminus) were microinjected at 450 ng/µl. cRNAs were synthesized using the T7 mMessage mMachine Kit (Ambion, cat# AM1340) and purified using the MEGAclear Kit (ThermoFisher, cat# AM1908) following the manufacturer’s protocols.

BUB1与EGFP在N端）以450 ng/µl的浓度进行了显微注射。cRNA使用T7 mMessage mMachine试剂盒（Ambion，货号# AM1340）合成，并按照制造商的方案使用MEGAclear试剂盒（ThermoFisher，货号# AM1908）进行纯化。

To visualize PMsat using the dCas9 technique, dCas9-EGFP cRNA (dead Cas9 with EGFP at the C-terminus, gift from Michael A. Lampson, 800 ng/µl) or dCas9-mCherry cRNA (dead Cas9 with mCherry at the C-terminus, gift from Michael A. Lampson, 800 ng/µl) was mixed with a cocktail of three sgRNAs that target PMsat sequences (PMsat 80, 5′-TAGATATGCCCCGTTTGTGT-3′; PMsat 223, 5′-TTACACTTAGTTGAGGCAAA-3′; PMsat 310, 5′-TCACGATAAACGTGACAAAT-3′; 150 ng/µl each) for microinjection.

为了使用dCas9技术可视化PMsat，将dCas9-EGFP cRNA（C端带有EGFP的失活Cas9，由Michael A. Lampson赠送，800 ng/µl）或dCas9-mCherry cRNA（C端带有mCherry的失活Cas9，由Michael A. Lampson赠送，800 ng/µl）与靶向PMsat序列的三种sgRNA混合物（PMsat 80，5′-TAGATATGCCCCGTTTGTGT-3′；PMsat 223，5′-TTACACTTAGTTGAGGCAAA-3′；PMsat 310，5′-TCACGATAAACGTGACAAAT-3′；各150 ng/µl）用于显微注射。

sgRNAs target part of PMsat consensus sequence that is conserved between .

sgRNAs 靶向 PMsat 共有序列中在两者之间保守的部分。

P. maniculatus

鹿鼠属

and

和

P. polionotus

. The sgRNAs were synthesized using GeneArt Precision gRNA Synthesis Kit (Thermo Fisher scientific, cat# A29377).

sgRNA使用GeneArt Precision gRNA合成试剂盒（Thermo Fisher scientific，货号# A29377）进行合成。

To Trim-Away REC8, mCherry-Trim21 cRNA (

为了修剪掉REC8，mCherry-Trim21 cRNA (

M. musculus domesticus

家鼠

Trim21 fused with mCherry at the C-terminus, Addgene cat# 105522) at 800 ng/µl and normal rabbit IgG (Sigma-Aldrich, cat# 12-370) or anti-REC8 antibody at 0.2 mg/ml (Invitrogen, cat# pa5-66964) were co-microinjected at the GV stage

在GV阶段，将C端与mCherry融合的Trim21（Addgene编号105522，浓度为800 ng/µl）和正常兔IgG（Sigma-Aldrich，编号12-370）或抗REC8抗体（Invitrogen，编号pa5-66964，浓度为0.2 mg/ml）共显微注射。

. REC8 degradation is specific to meiosis II likely due to the lower expression of TRIM21-mCherry in meiosis I (Fig.

REC8降解特异性地发生在减数分裂II，可能是由于减数分裂I中TRIM21-mCherry的表达较低（图。

and Supplementary Fig.

和补充图。

）。

Immunostaining of whole-mount cells and chromosome spreads

免疫染色全装细胞和染色体铺片

For whole-mount oocyte staining, meiosis I and II oocytes were fixed in freshly prepared 2% paraformaldehyde (Electron Microscopy Sciences, cat# 15710) in 1× PBS (Quality Biological, cat# 119-069-101) with 0.1% Triton X-100 (Millipore, cat# TX1568-1) for 20 min at room temperature, permeabilized in 1× PBS with 0.1% Triton X-100 for 15 min at room temperature, placed in the blocking solution (0.3% BSA (Fisher bioreagents, cat# BP1600-100) and 0.01% Tween-20 (Thermo Fisher Scientific, cat# J20605-AP) in 1× PBS) overnight at 4 °C, incubated 2 h with primary antibodies at room temperature, washed three times for 10 min with the blocking solution, incubated 1 h with secondary antibodies at room temperature, washed three times for 10 min in the blocking solution, and mounted on microscope slides with the Antifade Mounting Medium with DAPI (Vector Laboratories, cat# H-1200)..

对于整个卵母细胞染色，减数分裂I和II期的卵母细胞在新鲜配制的2%多聚甲醛（电子显微镜科学公司，目录号15710）中固定，该溶液以1× PBS（Quality Biological，目录号119-069-101）配制并含有0.1% Triton X-100（Millipore，目录号TX1568-1），室温下固定20分钟；然后在含0.1% Triton X-100的1× PBS中于室温下透化15分钟；随后置于封闭液（含0.3% BSA（Fisher生物试剂，目录号BP1600-100）和0.01% Tween-20（Thermo Fisher Scientific，目录号J20605-AP）的1× PBS溶液）中，4°C下孵育过夜；室温下与一抗孵育2小时；用封闭液洗涤3次，每次10分钟；室温下与二抗孵育1小时；再用封闭液洗涤3次，每次10分钟；最后使用含DAPI的抗褪色封片介质（Vector Laboratories，目录号H-1200）将样品封片于显微镜载玻片上。

For oocyte chromosome spreads, zona pellucida was removed from oocytes using Acidic Tyrode’s Solution (Millipore, cat# MR-004-D), and then the oocytes were transferred back to M2 or M16 media and cultured at 37 °C in a humidified atmosphere with 5% CO

对于卵母细胞染色体铺片，使用酸性泰罗德溶液（Millipore，产品编号 MR-004-D）去除卵母细胞的透明带，然后将卵母细胞转移回 M2 或 M16 培养基，并在 37°C 的湿润环境中以含 5% CO2 的条件进行培养。

for 30 min to 1 h to allow oocytes to recover. Subsequently, oocytes were fixed with 1% paraformaldehyde, 0.15% Triton X-100, and 3 mM DTT (Sigma, cat# 43815). After the oocytes burst on the microscope slide, the slides were placed in a closed humidified chamber and incubated overnight at room temperature to allow the chromatin to adhere to the slide.

将卵母细胞恢复30分钟至1小时。随后，用1%多聚甲醛、0.15% Triton X-100和3 mM DTT（Sigma，货号# 43815）固定卵母细胞。卵母细胞在显微镜载玻片上破裂后，将载玻片置于封闭的湿润腔室中，并在室温下孵育过夜，以使染色质附着在载玻片上。

The following day, the slides were air-dried completely and then stored in the freezer until immunostaining (see above)..

第二天，将玻片完全风干，然后存放在冰箱中直至进行免疫染色（见上文）。

Chromosome spread and whole-mount immunostaining for granulosa cells and bone marrow cells were performed as described above.

染色体铺展和全装免疫染色对于颗粒细胞和骨髓细胞的操作如上所述进行。

The following primary antibodies were used at the indicated delusions for both oocytes and somatic cells: rabbit anti-mouse REC8 (1:200, gift from Michael A. Lampson), mouse anti-human PP2A C subunit (1:100, EMD Millipore, cat# 05-421-AF488), rabbit anti-human Survivin (1:100, Cell Signaling Technology.

以下初级抗体在卵母细胞和体细胞中以指定的稀释比例使用：兔抗小鼠 REC8（1:200，Michael A. Lampson 赠送），小鼠抗人 PP2A C 亚基（1:100，EMD Millipore，货号# 05-421-AF488），兔抗人 Survivin（1:100，Cell Signaling Technology）。

cat# 2808), rabbit anti-human phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198), pAurora (1:100, Cell Signaling Technology, cat# 2914S), rabbit anti-human MCAK (1:1000, gift from Duane Compton), rabbit anti-histone H3-pT3 (1:100, Active Motif, cat# 39154), sheep polyclonal anti human-BUB1 antibody, SB1.3 (1:50, gift from Stephen Taylor), rabbit anti-histone H2A-pT120 (1:2000, Active motif, cat# 39391), mouse anti-human HEC1 (1:200, Santa Cruz, cat# sc-515550), CREST human autoantibody against centromere, ACA (1:100, Immunovision, cat# HCT-0100), goat anti-GFP antibody conjugated with Dylight488 (1:100, Rockland, cat# 600-141-215)..

cat# 2808)，兔抗人磷酸化-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198)，pAurora（1:100，Cell Signaling Technology，cat# 2914S），兔抗人MCAK（1:1000，Duane Compton馈赠），兔抗组蛋白H3-pT3（1:100，Active Motif，cat# 39154），羊多克隆抗人BUB1抗体，SB1.3（1:50，Stephen Taylor馈赠），兔抗组蛋白H2A-pT120（1:2000，Active motif，cat# 39391），小鼠抗人HEC1（1:200，Santa Cruz，cat# sc-515550），CREST人抗着丝粒自身抗体，ACA（1:100，Immunovision，cat# HCT-0100），山羊抗GFP抗体结合Dylight488（1:100，Rockland，cat# 600-141-215）。

Secondary antibodies were Alexa Fluor 488–conjugated donkey anti-rabbit (1:500, Invitrogen, cat# A21206) or donkey anti-goat (1:500, Invitrogen, cat# A11057), Alexa Fluor 568–conjugated goat anti-rabbit (1:500, Invitrogen, cat# A10042), or Alexa Fluor 647–conjugated goat anti-human (1:500, Invitrogen, cat# A21445)..

次级抗体为Alexa Fluor 488标记的驴抗兔（1:500，Invitrogen，货号A21206）或驴抗山羊（1:500，Invitrogen，货号A11057），Alexa Fluor 568标记的山羊抗兔（1:500，Invitrogen，货号A10042），或Alexa Fluor 647标记的山羊抗人（1:500，Invitrogen，货号A21445）。

Oligopaint design and oligopaint FISH of mitotic cells

寡聚体探针设计与有丝分裂细胞的寡聚体探针荧光原位杂交

Oligopaints were designed utilizing a modified version of the Oligominer pipeline, as previously described

Oligopaints 是利用经过修改的 Oligominer 管道设计的，如前所述。

. In brief, PMsat sequences, obtained from NCBI and spanning 340 bp, served as the foundation, and Bowtie2 was employed to identify oligos that uniquely mapped to the PMsat locus, utilizing the --very-sensitive-local alignment parameters. Oligo primers used to label PMsat: 5′-TTGGACTGAAGAGAAGCTCCTG-3′ and 5′-TGGGAACAGACGCGAGTG-3′..

简而言之，从NCBI获取的跨越340 bp的PMsat序列作为基础，使用Bowtie2通过--very-sensitive-local比对参数鉴定了唯一映射到PMsat位点的寡核苷酸。用于标记PMsat的寡核苷酸引物为：5′-TTGGACTGAAGAGAAGCTCCTG-3′和5′-TGGGAACAGACGCGAGTG-3′。

To label PMsat with oligopaint probes, cells were fixed in freshly prepared 2% paraformaldehyde (Thermo Fisher Scientific, cat# 28908) in 1× PBS (Quality Biological, cat# 119-069-101) for 20 min at room temperature. Subsequently, fixed cells underwent washing in a blocking solution (0.3% BSA (Fisher Bioreagents, cat# BP1600-100) and 0.01% Tween (Thermo Fisher Scientific, cat# J20605.AP) in 1× PBS.

为了用寡核苷酸探针标记PMsat，细胞在新鲜配制的2%多聚甲醛（Thermo Fisher Scientific，货号# 28908）中于1× PBS（Quality Biological，货号# 119-069-101）固定20分钟，室温下进行。随后，固定的细胞用封闭溶液（含0.3% BSA（Fisher Bioreagents，货号# BP1600-100）和0.01% Tween（Thermo Fisher Scientific，货号# J20605.AP）的1× PBS）进行洗涤。

The cells were permeabilized in 1× PBS with 0.1% Triton X-100 (Sigma-Aldrich, cat# TX1568-1) for 15 min at room temperature before returning to the blocking solution..

细胞在室温下用含 0.1% Triton X-100（Sigma-Aldrich，目录号 TX1568-1）的 1× PBS 渗透 15 分钟，然后回到封闭溶液中。

After immunostaining in the glass bottom chamber slide (Lab-Tek, cat# 155411) (see the previous section), the cells were then fixed a second time with 2% paraformaldehyde in 1x PBS for 10 min at room temperature. Slides were then washed (in coplin jars) with 1× PBS three times for 5 min at room temperature.

在玻璃底腔室玻片（Lab-Tek，产品编号155411）中进行免疫染色后（见前一节），细胞再用2%多聚甲醛在1倍PBS中于室温下固定10分钟。然后将玻片在室温下用1倍PBS洗涤三次，每次5分钟（使用Coplin染色缸）。

Subsequently, a primary oligopaint mix was added to each chamber well, and the chamber was sealed with parafilm. The primary oligopaint mix was composed of 100 pmol of each oligopaint, 1.5 µl of 25 µM dNTPs (New England BioLabs, cat# N0446S), 1 µl molecular grade H.

随后，向每个腔室孔中加入初级寡核苷酸混合物，并用封口膜密封腔室。初级寡核苷酸混合物由每种寡核苷酸100 pmol、1.5 µl 25 µM dNTPs（New England BioLabs，货号# N0446S）、1 µl分子级H组成。

O, 12.5 µl formamide, 4 µl PVSA (Sigma-Aldrich, cat# 278424), 1 µl RNase A (VWR Life Science, cat# E866-5ML), and 6.25 µl DNA hybridization buffer (4 g Dextran sulfate sodium salt (Sigma-Aldrich, cat# D8906-100G), 40 µl Tween, 4 ml 20× SSC, PVSA up to 10 ml), per reaction. After adding primary oligopaint mix, slides were heated to 85 °C on a metal block for 2.5 min and immediately transferred to a 37 °C humidified incubator for an overnight incubation.

每反应加入 12.5 µl 甲酰胺、4 µl PVSA（Sigma-Aldrich，目录号 278424）、1 µl RNA酶 A（VWR Life Science，目录号 E866-5ML）和 6.25 µl DNA 杂交缓冲液（4 g 葡聚糖硫酸钠盐（Sigma-Aldrich，目录号 D8906-100G）、40 µl Tween、4 ml 20× SSC，用 PVSA 补足至 10 ml）。加入主要寡核苷酸混合物后，将玻片在金属块上加热至 85°C 持续 2.5 分钟，然后立即转移到 37°C 的湿润培养箱中进行过夜孵育。

The following day, parafilm was removed and the slides were washed (in coplin jars) in 2× SSCT for 15 min at 60 °C, in 2× SSCT for 15 min at room temperature, and in 0.2× SSC for 10 min at room temperature. A secondary oligopaint mix was added to each chamber well, and the chamber was sealed with parafilm.

第二天，移除封口膜后，将载玻片（置于Coplin染色缸中）在60°C下用2× SSCT洗涤15分钟，在室温下用2× SSCT洗涤15分钟，并在室温下用0.2× SSC洗涤10分钟。随后向每个腔室孔中加入二级Oligopaint混合物，并用封口膜密封腔室。

The secondary oligopaint mix was composed of 10 pmol of each secondary oligo (IDT, custom synthesized), 6.25 µl DNA hybridization buffer, 12.5 µl formamide, and H.

次级寡核苷酸探针混合物由每种次级寡核苷酸（IDT，定制合成）10 pmol、6.25 µl DNA杂交缓冲液、12.5 µl甲酰胺和H组成。

O up to 25 µl, per reaction. Slides were then transferred to a 37 °C humidified incubator for 2 h. Subsequently, slides were washed in 2× SSCT for 15 min at 60 °C, in 2× SSCT for 15 min at room temperature, and in 0.2× SSC for 10 min at room temperature. A drop of Prolong Diamond Antifade Mountant with DAPI (Invitrogen, cat# P36966) was added to each chamber well..

每反应加入量为 25 µl。然后将玻片转移到 37°C 的湿润培养箱中孵育 2 小时。随后，将玻片在 60°C 的 2× SSCT 中洗涤 15 分钟，在室温下的 2× SSCT 中洗涤 15 分钟，并在室温下的 0.2× SSC 中洗涤 10 分钟。在每个腔室孔中加入一滴含 DAPI 的 Prolong Diamond 抗淬灭封片剂（Invitrogen，货号 P36966）。

Confocal microscopy and image analysis

共聚焦显微镜和图像分析

Fixed oocytes, bone marrow cells, and granulosa cells were imaged using a Nikon Eclipse Ti microscope. The microscope was equipped with a 100×/1.40 NA oil-immersion objective lens, a CSU-W1 spinning disk confocal scanner by Yokogawa, an ORCA Fusion Digital CMOS camera from Hamamatsu Photonics, and controlled laser lines at 405 nm, 488 nm, 561 nm, and 640 nm via NIS-Elements imaging software by Nikon.

固定卵母细胞、骨髓细胞和颗粒细胞使用尼康Eclipse Ti显微镜进行成像。该显微镜配备有100×/1.40 NA油浸物镜、横河CSU-W1旋转盘共聚焦扫描仪、滨松光电的ORCA Fusion数字CMOS相机，并通过尼康的NIS-Elements成像软件控制405 nm、488 nm、561 nm和640 nm激光线。

Confocal images were captured as Z-stacks at 0.3 µm intervals, and these images were presented as maximum intensity Z-projections unless specified in the figure legend..

共聚焦图像以0.3微米的间隔捕获为Z栈，并且除非图例中另有说明，这些图像以最大强度Z投影的形式呈现。

For image analysis, Fiji/ImageJ (NIH) software was employed. First, optical slices containing chromosomes were combined to generate sum intensity Z-projections for subsequent pixel intensity quantifications. Signal intensities on the entire chromosome (Survivin and H2A-pT121) were quantified by creating masking images using the DAPI staining.

对于图像分析，使用了Fiji/ImageJ（NIH）软件。首先，将包含染色体的光学切片合并以生成用于后续像素强度量化的总强度Z投影。通过使用DAPI染色创建掩膜图像，对整个染色体上的信号强度（Survivin和H2A-pT121）进行了量化。

Signal intensities were integrated over each slice after the background signal subtraction. To specifically quantify centromeric signal intensities (PP2A, Survivin, pAurora, MCAK, BUB1, H2A-pT121, and H3-pT3), ellipses were delineated around PMsat or the kinetochore (based on the HEC1 staining) on each chromosome.

在背景信号减去后，对每一片层的信号强度进行了积分。为了特别量化着丝粒信号强度（PP2A、Survivin、pAurora、MCAK、BUB1、H2A-pT121 和 H3-pT3），在每个染色体上根据HEC1染色围绕PMsat或动粒划定了椭圆。

Signal intensities were then quantified within each ellipse after the background signal subtraction..

然后在每个椭圆内对信号强度进行量化，同时减去背景信号。

Statistics and reproducibility

统计与可重复性

Data points were pooled from three independent experiments in most experiments, and the exact number of independent experiments for each experimental group is listed in Supplementary Table

在大多数实验中，数据点是从三个独立实验中汇总的，每个实验组的独立实验的确切次数列在补充表中。

. Data analysis was performed using Microsoft Excel and GraphPad Prism 10. Scattered plots and line graphs were created with GraphPad Prism 10. unpaired two-tailed Mann-Whitney test and unpaired two-sided t-test were used for statistical analysis unless specified in the figure legend, and the exact .

数据分析使用 Microsoft Excel 和 GraphPad Prism 10 进行。散点图和折线图使用 GraphPad Prism 10 创建。除非图例中另有说明，统计分析采用非配对双尾 Mann-Whitney 检验和非配对双侧 t 检验，并给出精确的 p 值。

values are shown in each figure. The sample size was chosen based on current practices in the field. Randomization is built into the experiments because each animal was chosen from a different litter and mating pair and no data was excluded and all cells were imaged at random.

每个图中都显示了数值。样本大小是根据该领域的现行惯例选择的。实验中包含了随机化，因为每只动物都来自不同的窝和交配对，且没有数据被排除，所有细胞都是随机成像的。

Reporting summary

报告摘要

Further information on research design is available in the

有关研究设计的更多信息，请参见

Nature Portfolio Reporting Summary

《自然》系列报告摘要

linked to this article.

与本文相关联。

Data availability

数据可用性

Raw data files for the DNA sequencing analysis have been deposited in the NCBI Sequence Read Archive (SRA) and are available under project accession number SRA:

DNA测序分析的原始数据文件已存入NCBI序列读取档案库（SRA），并可在项目登录号SRA下获取：

PRJNA1196496

. Other data required to reproduce the results in the current study are available at Figshare [

。重现当前研究结果所需的其他数据可在 Figshare [ 获取

https://doi.org/10.25444/nhlbi.28001618

Source data

源数据

are provided with this paper.

随本文一起提供。

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Acknowledgements

致谢

We thank Alexander E. Kelly for comments on the manuscript, Rachel J. O’Neil for discussion and PMsat reagents, Duane Compton for the MCAK antibody, Michael A. Lampson for the REC8 antibody and the pIVT-dCas9-EGFP and pIVT-dCas9-mCherry plasmids, Stephen Taylor for the BUB1 antibody, R. Zaak Walton for establishing and maintaining .

我们感谢亚历山大·E·凯利对稿件的评论，雷切尔·J·奥尼尔的讨论和PMsat试剂，杜安·康普顿提供的MCAK抗体，迈克尔·A·兰姆森提供的REC8抗体以及pIVT-dCas9-EGFP和pIVT-dCas9-mCherry质粒，斯蒂芬·泰勒提供的BUB1抗体，R·扎克·沃尔顿的建立和维护。

Peromyscus

鹿鼠属

mouse colonies, and the Akera lab members for discussion. This work is supported by the Intramural Programs of National Heart, Lung, and Blood Institute (1ZIAHL006249) (T.A.) and the

小鼠群体，以及Akera实验室成员的讨论。这项工作得到了国家心肺血液研究所（1ZIAHL006249）（T.A.）和

Eunice Kennedy Shriver

尤妮斯·肯尼迪·施赖弗

National Institute of Child Health and Human Development (1ZIAHD008933) (T.S.M.) at the National Institutes of Health (NIH).

美国国立卫生研究院（NIH）的国家儿童健康与人类发展研究所（1ZIAHD008933）（T.S.M.）。

Funding

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Open access funding provided by the National Institutes of Health.

由美国国立卫生研究院提供开放获取资助。

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Authors and Affiliations

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Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

美国马里兰州贝塞斯达国立卫生研究院，国家心肺血液研究所，细胞与发育生物学中心

Bo Pan & Takashi Akera

潘博 & 赤井孝

The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

美国马里兰州贝塞斯达国立卫生研究院尤尼斯·肯尼迪·施赖弗国家儿童健康与人类发展研究所

Melania Bruno & Todd S. Macfarlan

梅拉尼娅·布鲁诺和托德·S·麦克法兰

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Bo Pan

潘博

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Takashi Akera

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Contributions

贡献

Conceptualization, T.A.; Methodology, B.P. and M.B.; Investigation, T.A., B.P., M.B., and T.S.M.; Writing—Original Draft, B.P.; Writing—Review & Editing, T.A., B.P., M.B., and T.S.M.; Funding Acquisition, T.A. and T.S.M.; Resources, T.A. and T.S.M.; Supervision, T.A. and T.S.M.

概念化，T.A.；方法论，B.P. 和 M.B.；调查，T.A.、B.P.、M.B. 和 T.S.M.；撰写—原稿，B.P.；撰写—审核与编辑，T.A.、B.P.、M.B. 和 T.S.M.；资金获取，T.A. 和 T.S.M.；资源，T.A. 和 T.S.M.；监督，T.A. 和 T.S.M.

Corresponding author

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Correspondence to

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Takashi Akera

高桥明良

。

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Nature Communications

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thanks Stefan Heidmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

感谢Stefan Heidmann和另一位匿名评审员对本工作同行评审的贡献。同行评审文件可供查阅。

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Pan, B., Bruno, M., Macfarlan, T.S.