Phylogenetic and Expression Analysis of CENH3 and APOLLO Genes in Sexual and Apomictic Boechera Species

Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO (Aspartate Glutamate Aspartate Aspartate histidine exonuclease) is one of the very few described genes associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Also, we characterize the structure of CENH3, study expression levels of APOLLO and CENH3 in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of before and after fertilization. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes.

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Phylogenetic and Expression Analysis of CENH3 and APOLLO Genes in Sexual and Apomictic Boechera Species

10.20944/preprints202101.0320.v1 ◽

2021 ◽

Author(s):

Evgeny Bakin ◽

Fatih Sezer ◽

Irem Kilic ◽

Aslıhan Özbilen ◽

Mike Rayko ◽

...

Keyword(s):

Chromosome Segregation ◽

Genetic Regulation ◽

Single Copy ◽

Egg Cell ◽

Expression Levels ◽

Histone H3 Variant ◽

Before And After ◽

Spindle Microtubules ◽

Mitosis And Meiosis

Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO (Aspartate Glutamate Aspartate Aspartate histidine exonuclease) is the only described gene associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Also, in this research we characterize the structure of CENH3, study expression levels of CENH3 and APOLLO in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of before and after fertilization. At the premeiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, it decreased in both species by the time of meiosis and increased after fertilization. By 1 DAP CENH3 expression started dropping in sexual B. stricta siliques and kept increasing in apomictic B. divaricarpa ones. That might indicate to a role of CENH3 in apomictic development in Boechera species. The expression levels of APOLLO also sharply decreased by the time of meiosis in gynoecium of both species; however, by 3 DAP, the level of APOLLO expression in siliques of apomicts was almost 1.5 times higher than that of the sexuals. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes.

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Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

Open Biology ◽

10.1098/rsob.150236 ◽

2016 ◽

Vol 6 (2) ◽

pp. 150236 ◽

Cited By ~ 24

Author(s):

Yahui Liu ◽

Arsen Petrovic ◽

Pascaline Rombaut ◽

Shyamal Mosalaganti ◽

Jenny Keller ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Chromosome Segregation ◽

Functional Organization ◽

Histone H3 Variant ◽

Centromeric Protein ◽

Spindle Microtubules ◽

Specialized Region ◽

Structural Methods ◽

Mitosis And Meiosis ◽

Shed Light

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.

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Identification of four unconventional kinetoplastid kinetochore proteins KKT22–25 in Trypanosoma brucei

Open Biology ◽

10.1098/rsob.190236 ◽

2019 ◽

Vol 9 (12) ◽

pp. 190236 ◽

Cited By ~ 6

Author(s):

Olga O. Nerusheva ◽

Patryk Ludzia ◽

Bungo Akiyoshi

Keyword(s):

Trypanosoma Brucei ◽

Chromosome Segregation ◽

Rna Binding ◽

Affinity Purification ◽

Potential Interaction ◽

Interacting Proteins ◽

Interaction Partners ◽

Spindle Microtubules ◽

Acetyltransferase Domain ◽

Mitosis And Meiosis

The kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1–20). In addition, 12 proteins (KKT-interacting proteins 1–12, KKIP1–12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22–25, in Trypanosoma brucei . KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N -acetyltransferase domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.

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ConnectingGCN5’s centromeric SAGA to the mitotic tension-sensing checkpoint

Molecular Biology of the Cell ◽

10.1091/mbc.e17-12-0701 ◽

2018 ◽

Vol 29 (18) ◽

pp. 2201-2212 ◽

Cited By ~ 1

Author(s):

Emily L. Petty ◽

Masha Evpak ◽

Lorraine Pillus

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Spindle Assembly Checkpoint ◽

Binding Function ◽

Chromatid Cohesion ◽

Histone H3 Variant ◽

Spindle Microtubules ◽

Low Tension ◽

Centromere Histone H3 ◽

Segregation Of Chromosomes

Multiple interdependent mechanisms ensure faithful segregation of chromosomes during cell division. Among these, the spindle assembly checkpoint monitors attachment of spindle microtubules to the centromere of each chromosome, whereas the tension-sensing checkpoint monitors the opposing forces between sister chromatid centromeres for proper biorientation. We report here a new function for the deeply conserved Gcn5 acetyltransferase in the centromeric localization of Rts1, a key player in the tension-sensing checkpoint. Rts1 is a regulatory component of protein phopshatase 2A, a near universal phosphatase complex, which is recruited to centromeres by the Shugoshin (Sgo) checkpoint component under low-tension conditions to maintain sister chromatid cohesion. We report that loss of Gcn5 disrupts centromeric localization of Rts1. Increased RTS1 dosage robustly suppresses gcn5∆ cell cycle and chromosome segregation defects, including restoration of Rts1 to centromeres. Sgo1’s Rts1-binding function also plays a key role in RTS1 dosage suppression of gcn5∆ phenotypes. Notably, we have identified residues of the centromere histone H3 variant Cse4 that function in these chromosome segregation-related roles of RTS1. Together, these findings expand the understanding of the mechanistic roles of Gcn5 and Cse4 in chromosome segregation.

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Decoding the centromeric nucleosome through CENP-N

eLife ◽

10.7554/elife.33442 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 44

Author(s):

Satyakrishna Pentakota ◽

Keda Zhou ◽

Charlotte Smith ◽

Stefano Maffini ◽

Arsen Petrovic ◽

...

Keyword(s):

Chromosome Segregation ◽

Double Helix ◽

Structural Basis ◽

Binding Motif ◽

Kinetochore Assembly ◽

Nucleosomal Dna ◽

Chromosome Domains ◽

Histone H3 Variant ◽

Spindle Microtubules ◽

Dna Double Helix

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

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The ctf13-30/CTF13 Genomic Haploinsufficiency Modifier Screen Identifies the Yeast Chromatin Remodeling Complex RSC, Which Is Required for the Establishment of Sister Chromatid Cohesion

Molecular and Cellular Biology ◽

10.1128/mcb.24.3.1232-1244.2003 ◽

2004 ◽

Vol 24 (3) ◽

pp. 1232-1244 ◽

Cited By ~ 93

Author(s):

Kristin K. Baetz ◽

Nevan J. Krogan ◽

Andrew Emili ◽

Jack Greenblatt ◽

Philip Hieter

Keyword(s):

Chromatin Remodeling ◽

Chromosome Segregation ◽

High Fidelity ◽

Chromosome Transmission ◽

New Genes ◽

Sister Chromatids ◽

Chromatin Remodeling Complex ◽

Chromatid Cohesion ◽

Spindle Microtubules ◽

Mitosis And Meiosis

ABSTRACT The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules. To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point. We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation. One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex. rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected. We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion. In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified. Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity.

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Embryo development in Carica papaya Linn

10.1101/2021.03.11.434975 ◽

2021 ◽

Author(s):

Miguel Acevedo-Benavides ◽

Pablo Bolaños-Villegas

Keyword(s):

Cell Fate ◽

Female Gametophyte ◽

Carica Papaya ◽

Genetic Regulation ◽

Draft Genome ◽

Embryo Sac ◽

Egg Cell ◽

Parental Line ◽

Before And After ◽

Autonomous Development

ABSTRACTPapaya (Carica papaya Linn.) is a tropical plant whose draft genome has been sequenced. Papaya produces large fruits rich in vitamins A and C and is an important cash crop in developing countries. Nonetheless, little is known about how the female gametophyte develops, how it is fertilized and how it develops into a mature seed containing an embryo and an endosperm. The Papaya female gametophyte displays a Polygonum-type architecture consisting of two synergid cells, an egg cell, a central cell, and three antipodal cells. Reports are available of the presumed existence of varieties in which cross fertilization is bypassed and autonomous development of embryos occurs (e.g., apomixis). In this study, we analyzed the development of female gametophytes in a commercial Hawaiian parental line and in the presumed apomictic Costa Rican line L1. Samples were collected before and after anthesis to compare the overall structure, size and transcriptional patterns of several genes that may be involved in egg and endosperm cell fate and proliferation. These genes were the putative papaya homologs of ARGONAUTE9 (AGO9), MEDEA (MEA), RETINOBLASTOMA RELATED-1 (RBR1), and SLOW WALKER-1 (SWA1). Our results suggest that its feasible to identify the contour of structural features of Polygonum-type development, and that in bagged female flowers of line L1 we might have observed autonomous development of embryo-like structures. Possible downregulation of papaya homologs for AGO9, MEA, RBR1 and SWA1 was observed in embryo sacs from line L1 before and after anthesis, which may suggest a tentative link between suspected apomixis and transcriptional downregulation of genes for RNA-directed DNA methylation, histone remodelers, and rRNA processing. Most notably, the large size of the papaya embryo sac suggests that it could be a cytological alternative to Arabidopsis thaliana for study. Significant variation in embryo sac size was observed between the varieties under study, suggesting wide differences in the genetic regulation of anatomical features.

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Establishing correct kinetochore-microtubule attachments in mitosis and meiosis

Essays in Biochemistry ◽

10.1042/ebc20190072 ◽

2020 ◽

Vol 64 (2) ◽

pp. 277-287

Author(s):

Gisela Cairo ◽

Soni Lacefield

Keyword(s):

Error Correction ◽

Chromosome Segregation ◽

Germ Cells ◽

Chromosomal Localization ◽

Aurora B ◽

Molecular Pathways ◽

Human Cell Lines ◽

Chromosomal Passenger ◽

Spindle Microtubules ◽

Mitosis And Meiosis

Abstract Faithful chromosome segregation in mitosis and meiosis requires that chromosomes properly attach to spindle microtubules. Initial kinetochore-microtubule attachments are often incorrect and rely on error correction mechanisms to release improper attachments, allowing the formation of new attachments. Aurora B kinase and, in mammalian germ cells, Aurora C kinase function as the enzymatic component of the Chromosomal Passenger Complex (CPC), which localizes to the inner centromere/kinetochore and phosphorylates kinetochore proteins for microtubule release during error correction. In this review, we discuss recent findings of the molecular pathways that regulate the chromosomal localization of Aurora B and C kinases in human cell lines, mice, fission yeast, and budding yeast. We also discuss differences in the importance of localization pathways between mitosis and meiosis.

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The centromere comes into focus: from CENP-A nucleosomes to kinetochore connections with the spindle

Open Biology ◽

10.1098/rsob.200051 ◽

2020 ◽

Vol 10 (6) ◽

pp. 200051 ◽

Cited By ~ 5

Author(s):

Kathryn Kixmoeller ◽

Praveen Kumar Allu ◽

Ben E. Black

Keyword(s):

Chromosome Segregation ◽

Molecular Complexes ◽

Molecular Models ◽

Chromosomal Locus ◽

Centromeric Chromatin ◽

Eukaryotic Chromosome ◽

The Core ◽

Histone H3 Variant ◽

Spindle Microtubules ◽

Insight Into

Eukaryotic chromosome segregation relies upon specific connections from DNA to the microtubule-based spindle that forms at cell division. The chromosomal locus that directs this process is the centromere, where a structure called the kinetochore forms upon entry into mitosis. Recent crystallography and single-particle electron microscopy have provided unprecedented high-resolution views of the molecular complexes involved in this process. The centromere is epigenetically specified by nucleosomes harbouring a histone H3 variant, CENP-A, and we review recent progress on how it differentiates centromeric chromatin from the rest of the chromosome, the biochemical pathway that mediates its assembly and how two non-histone components of the centromere specifically recognize CENP-A nucleosomes. The core centromeric nucleosome complex (CCNC) is required to recruit a 16-subunit complex termed the constitutive centromere associated network (CCAN), and we highlight recent structures reported of the budding yeast CCAN. Finally, the structures of multiple modular sub-complexes of the kinetochore have been solved at near-atomic resolution, providing insight into how connections are made to the CCAN on one end and to the spindle microtubules on the other. One can now build molecular models from the DNA through to the physical connections to microtubules.

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Identification of four unconventional kinetoplastid kinetochore proteins KKT22–25 in Trypanosoma brucei

10.1101/785170 ◽

2019 ◽

Cited By ~ 1

Author(s):

Olga O. Nerusheva ◽

Patryk Ludzia ◽

Bungo Akiyoshi

Keyword(s):

Trypanosoma Brucei ◽

Chromosome Segregation ◽

Protein Complex ◽

Rna Binding ◽

Affinity Purification ◽

Potential Interaction ◽

S Phase ◽

Interaction Partners ◽

Spindle Microtubules ◽

Mitosis And Meiosis

SummaryThe kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1–20). In addition, twelve proteins (KKIP1–12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22–25, in Trypanosoma brucei. KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N-acetyltransferase (GNAT) domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.

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