Shihori Yokobayashi

Cohesin relocation from sites of chromosomal loading to places of convergent transcription.

Sister chromatids, the products of eukaryotic DNA replication, are held together by the chromosomal cohesin complex after their synthesis. This allows the spindle in mitosis to recognize pairs of replication products for segregation into opposite directions. Cohesin forms large protein rings that may bind DNA strands by encircling them, but the characterization of cohesin binding to chromosomes in vivo has remained vague. We have performed high resolution analysis of cohesin association along budding yeast chromosomes III–VI. Cohesin localizes almost exclusively between genes that are transcribed in converging directions. We find that active transcription positions cohesin at these sites, not the underlying DNA sequence. Cohesin is initially loaded onto chromosomes at separate places, marked by the Scc2/Scc4 cohesin loading complex, from where it appears to slide to its more permanent locations. But even after sister chromatid cohesion is established, changes in transcription lead to repositioning of cohesin. Thus the sites of cohesin binding and therefore probably sister chromatid cohesion, a key architectural feature of mitotic chromosomes, display surprising flexibility. Cohesin localization to places of convergent transcription is conserved in fission yeast, suggesting that it is a common feature of eukaryotic chromosomes.

Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast

Fission yeast centromeres, like those of higher eukaryotes, are composed of repeated DNA structures and associated heterochromatin protein complexes, that have a critical function in the faithful segregation of chromosomes during cell division. Cohesin protein complexes, which are essential for sister-chromatid cohesion and proper chromosome segregation, are enriched at centromeric repeats. We have identified a functional and physical link between heterochromatin and cohesin. We find that the preferential localization of cohesins at the centromeric repeats is dependent on Swi6, a conserved heterochromatin protein that is required for proper kinetochore function. Cohesin is also enriched at the mating-type heterochromatic region in a manner that depends on Swi6 and is required to preserve the genomic integrity of this locus. We provide evidence that a cohesin subunit Psc3 interacts with Swi6 and its mouse homologue HP1. These data define a conserved function of Swi6/HP1 in recruitment of cohesin to heterochromatic regions, promoting the proper segregation of chromosomes.

Polycomb Group Proteins Ezh2 and Rnf2 Direct Genomic Contraction and Imprinted Repression in Early Mouse Embryos

Genomic imprinting regulates parental-specific expression of particular genes and is required for normal mammalian development. How imprinting is established during development is, however, largely unknown. To address this question, we studied the mouse Kcnq1 imprinted cluster at which paternal-specific silencing depends on expression of the noncoding RNA Kcnq1ot1. We show that Kcnq1ot1 is expressed from the zygote stage onward and rapidly associates with chromatin marked by Polycomb group (PcG) proteins and repressive histone modifications, forming a discrete repressive nuclear compartment devoid of RNA polymerase II, a configuration also observed at the Igf2r imprinted cluster. In this compartment, the paternal Kcnq1 cluster exists in a three-dimensionally contracted state. In vivo the PcG proteins Ezh2 and Rnf2 are independently required for genomic contraction and imprinted silencing. We propose that the formation of a parental-specific higher-order chromatin organization renders imprint clusters competent for monoallelic silencing and assign a central role to PcG proteins in this process.

Pre-meiotic S phase is linked to reductional chromosome segregation and recombination.

Meiosis is initiated from G1 of the cell cycle and is characterized by a pre-meiotic S phase followed by two successive nuclear divisions. The first of these, meiosis I, differs from mitosis in having a reductional pattern of chromosome segregation. Here we show that meiosis can be initiated from G2 in fission yeast cells by ectopically activating the meiosis-inducing network. The subsequent meiosis I occurs without a pre-meiotic S phase and with decreased recombination, and exhibits a mitotic pattern of equational chromosome segregation. The subsequent meiosis II results in random chromosome segregation. This behaviour is similar to that observed in cells lacking the meiotic cohesin Rec8 (refs 3, 4), which becomes associated with chromosomes at G1/S phase, including the inner centromere, a region that is probably critical for sister-centromere orientation. If the expression of Rec8 is delayed to S phase/G2, then the centromeres behave equationally. We propose that the presence of Rec8 in chromatin is required at the pre-meiotic S phase to construct centromeres that behave reductionally and chromosome arms capable of a high level of recombination, and that this explains why meiosis is initiated from G1 of the cell cycle.

Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-alpha-associated protein Ctf4 is essential for chromatid disjunction during meiosis II

Cohesion between sister chromatids mediated by a multisubunit complex called cohesin is established during DNA replication and is essential for the orderly segregation of chromatids during anaphase. In budding yeast, a specialized replication factor C called RF-CCtf18/Dcc1/Ctf8 and the DNA-polymerase-α-associated protein Ctf4 are required to maintain sister-chromatid cohesion in cells arrested for long periods in mitosis. We show here that CTF8, CTF4 and a helicase encoded by CHL1 are required for efficient sister chromatid cohesion in unperturbed mitotic cells, and provide evidence that Chl1 functions during S-phase. We also show that, in contrast to mitosis, RF-CCtf18/Dcc1/Cft8, Ctf4 and Chl1 are essential for chromosome segregation during meiosis and for the viability of meiotic products. Our finding that cells deleted for CTF8, CTF4 or CHL1 undergo massive meiosis II non-disjunction suggests that the second meiotic division is particularly sensitive to cohesion defects. Using a functional as well as a cytological assay, we demonstrate that CTF8, CHL1 and CTF4 are essential for cohesion between sister centromeres during meiosis but dispensable for cohesins association with centromeric DNA. Our finding that mutants in fission yeast ctf18 and dcc1 have similar defects suggests that the involvement of the alternative RF-CCtf18/Dcc1/Ctf8 complex in sister chromatid cohesion might be highly conserved.

Cohesins Determine the Attachment Manner of Kinetochores to Spindle Microtubules at Meiosis I in Fission Yeast

ABSTRACT During mitosis, sister kinetochores attach to microtubules that extend to opposite spindle poles (bipolar attachment) and pull the chromatids apart at anaphase (equational segregation). A multisubunit complex called cohesin, including Rad21/Scc1, plays a crucial role in sister chromatid cohesion and equational segregation at mitosis. Meiosis I differs from mitosis in having a reductional pattern of chromosome segregation, in which sister kinetochores are attached to the same spindle (monopolar attachment). During meiosis, Rad21/Scc1 is largely replaced by its meiotic counterpart, Rec8. If Rec8 is inactivated in fission yeast, meiosis I is shifted from reductional to equational division. However, the reason rec8Δ cells undergo equational rather than random division has not been clarified; therefore, it has been unclear whether equational segregation is due to a loss of cohesin in general or to a loss of a specific requirement for Rec8. We report here that the equational segregation at meiosis I depends on substitutive Rad21, which relocates to the centromeres if Rec8 is absent. Moreover, we demonstrate that even if sufficient amounts of Rad21 are transferred to the centromeres at meiosis I, thereby establishing cohesion at the centromeres, rec8Δ cells never recover monopolar attachment but instead secure bipolar attachment. Thus, Rec8 and Rad21 define monopolar and bipolar attachment, respectively, at meiosis I. We conclude that cohesin is a crucial determinant of the attachment manner of kinetochores to the spindle microtubules at meiosis I in fission yeast.

PRC1 coordinates timing of sexual differentiation of female primordial germ cells

In mammals, sex differentiation of primordial germ cells (PGCs) is determined by extrinsic cues from the environment. In mouse female PGCs, expression of stimulated by retinoic acid gene 8 (Stra8) and meiosis are induced in response to retinoic acid provided from the mesonephroi. Given the widespread role of retinoic acid signalling during development, the molecular mechanisms that enable PGCs to express Stra8 and enter meiosis in a timely manner are unknown. Here we identify gene-dosage-dependent roles in PGC development for Ring1 and Rnf2, two central components of the Polycomb repressive complex 1 (PRC1). Both paralogues are essential for PGC development between days 10.5 and 11.5 of gestation. Rnf2 is subsequently required in female PGCs to maintain high levels of Oct4 (also known as Pou5f1) and Nanog expression, and to prevent premature induction of meiotic gene expression and entry into meiotic prophase. Chemical inhibition of retinoic acid signalling partially suppresses precocious Oct4 downregulation and Stra8 activation in Rnf2-deficient female PGCs. Chromatin immunoprecipitation analyses show that Stra8 is a direct target of PRC1 and PRC2 in PGCs. These data demonstrate the importance of PRC1 gene dosage in PGC development and in coordinating the timing of sex differentiation of female PGCs by antagonizing extrinsic retinoic acid signalling.

Meikin is a conserved regulator of meiosis-I-specific kinetochore function

The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.

Author Correction: Meikin is a conserved regulator of meiosis-I-specific kinetochore function

An Amendment to this Article has been published and is linked from the HTML version of this paper.