Role In Chromosome Segregation Research Articles

An extrinsic motor directs chromatin loop formation by cohesin.

The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation.

Topoisomerase I is an evolutionarily conserved key regulator for satellite DNA transcription

RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. We here show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

Coupling Kinesin Spindle Protein and Aurora B Inhibition with Apoptosis Induction Enhances Oral Cancer Cell Killing.

Many proteins regulating mitosis have emerged as targets for cancer therapy, including the kinesin spindle protein (KSP) and Aurora kinase B (AurB). KSP is crucial for proper spindle pole separation during mitosis, while AurB plays roles in chromosome segregation and cytokinesis. Agents targeting KSP and AurB selectively affect dividing cells and have shown significant activity in vitro. However, these drugs, despite advancing to clinical trials, often yield unsatisfactory outcomes as monotherapy, likely due to variable responses driven by cyclin B degradation and apoptosis signal accumulation networks. Accumulated data suggest that combining emerging antimitotics with various cytostatic drugs can enhance tumor-killing effects compared to monotherapy. Here, we investigated the impact of inhibiting anti-apoptotic signals with the BH3-mimetic Navitoclax in oral cancer cells treated with the selective KSP inhibitor, Ispinesib, or AurB inhibitor, Barasertib, aiming to potentiate cell death. The combination of BH3-mimetics with both KSP and AurB inhibitors synergistically induced substantial cell death, primarily through apoptosis. A mechanistic analysis underlying this synergistic activity, undertaken by live-cell imaging, is presented. Our data underscore the importance of combining BH3-mimetics with antimitotics in clinical trials to maximize their effectiveness.

The outer kinetochore components KNL-1 and Ndc80 complex regulate axon and neuronal cell body positioning in the C. elegans nervous system.

The KMN (Knl1/Mis12/Ndc80) network at the kinetochore, primarily known for its role in chromosome segregation, has been shown to be repurposed during neurodevelopment. Here, we investigate the underlying neuronal mechanism and show that the KMN network promotes the proper axonal organization within the C. elegans head nervous system. Postmitotic degradation of KNL-1, which acts as a scaffold for signaling and has microtubule-binding activities at the kinetochore, led to disorganized ganglia and aberrant placement and organization of axons in the nerve ring - an interconnected axonal network. Through gene-replacement approaches, we demonstrate that the signaling motifs within KNL-1, responsible for recruiting protein phosphatase 1, and activating the spindle assembly checkpoint are required for neurodevelopment. Interestingly, while the microtubule-binding activity is crucial to KMN's neuronal function, microtubule dynamics and organization were unaffected in the absence of KNL-1. Instead, the NDC-80 microtubule-binding mutant displayed notable defects in axon bundling during nerve ring formation, indicating its role in facilitating axon-axon contacts. Overall, these findings provide evidence for a noncanonical role for the KMN network in shaping the structure and connectivity of the nervous system in C. elegans during brain development.

Plant kinetochore complex: composition, function, and regulation.

The kinetochore complex, an important protein assembly situated on the centromere, plays a pivotal role in chromosome segregation during cell division. Like in animals and fungi, the plant kinetochore complex is important for maintaining chromosome stability, regulating microtubule attachment, executing error correction mechanisms, and participating in signaling pathways to ensure accurate chromosome segregation. This review summarizes the composition, function, and regulation of the plant kinetochore complex, emphasizing the interactions of kinetochore proteins with centromeric DNAs (cenDNAs) and RNAs (cenRNAs). Additionally, the applications of the centromeric histone H3 variant (the core kinetochore protein CENH3, first identified as CENP-A in mammals) in the generation of ploidy-variable plants and synthesis of plant artificial chromosomes (PACs) are discussed. The review serves as a comprehensive roadmap for researchers delving into plant kinetochore exploration, highlighting the potential of kinetochore proteins in driving technological innovations in synthetic genomics and plant biotechnology.

Aurora kinase B disruption suppresses pathological retinal angiogenesis by affecting cell cycle progression

PurposeThe detrimental effects of pathological angiogenesis on the visual function are indisputable. Within a prominent role in chromosome segregation and tumor progression, aurora kinase B (AURKB) assumes a prominent role. However, its role in pathological retinal angiogenesis remains unclear. This study explores this latent mechanism. MethodsTo inhibit AURKB expression, we designed specific small interfering RNAs targeting AURKB and transfected them into vascular endothelial cells. Barasertib was selected as the AURKB inhibitor. The anti-angiogenic effects of both AURKB siRNA and barasertib were assessed in vitro by cell proliferation, transwell migration, and tube formation. To evaluate the angiogentic effects of AURKB in vivo, neonatal mice were exposed to 75% oxygen followed by normoxic repositioning to establish an oxygen-induced retinopathy (OIR) model. Subsequently, phosphate-buffered saline and barasertib were administered into OIR mice via intravitreal injection. The effects of AURKB on cell cycle proteins were determined by western blot analysis. ResultsWe found that AURKB was overexpressed during pathological angiogenesis. AURKB siRNA and barasertib significantly inhibited endothelial cell proliferation, migration, and tube formation in vitro. Furthermore, AURKB inhibition attenuated retinal angiogenesis in the OIR model. A possible mechanism is the disruption of cell cycle by AURKB inhibition. ConclusionIn conclusion, AURKB significantly influenced pathological retinal angiogenesis, thereby presenting a promising therapeutic target in ocular neovascular diseases.

The kleisin subunit controls the function of C. elegans meiotic cohesins by determining the mode of DNA binding and differential regulation by SCC-2 and WAPL-1.

The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in Caenorhabditis elegans. How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin-DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes.

Evaluation of miRNA-200c and βIII tubulin as promising markers for clinical decision making in locally advanced breast cancer patients receiving taxanes therapy

BackgroundClinical management of locally advanced breast cancer (LABC) is a common clinical problem. Therefore, finding a good predictor for the efficacy of treatment is crucial. The miRNAs are small non-coding RNAs that play an important role in gene regulation of many biological processes. Class III tubulin (βIII tubulin) are dynamic fibrous cytoskeletal proteins that play a significant role in chromosome segregation during mitosis and meiosis and has been identified to participate in cell proliferation and cancer development. AimTo evaluate the role of miRNA-200c and βIII tubulin as promising markers for clinical decision making in locally advanced breast cancer patients receiving taxanes therapy. MethodsA retrospective study of paraffin sections of 143 female breast patients: 22 patients with benign breast diseases and 121 breast cancer patients receiving neoadjuvant taxane therapy divided to: 94 responder patients and 27 non responder patients. Patients were followed up from date of first diagnosis till end of the study. The expression level of miRNA 200c was evaluated using real time PCR and the expression level of βIII tubulin protein was immunohistochemically estimated. Results were correlated with response to neoadjuvant taxanes therapy. ResultsThe miRNA 200 c was significantly down regulated in patients responding to taxanes as compared with non-responders (p = 0.009). The βIII tubulin protein was significantly highly expressed in ER negative and PR negative breast cancer patients (p = 0.011, p = 0.053, respectively). It was significantly up regulated in triple negative and HER2 enriched (non luminal) cases and down regulated in luminal cases (p = 0.023). ConclusionThe results suggest miRNA 200c may be a predictive marker for response to neoadjuvant taxanes therapy in breast cancer patients, while βIII tubulin protein showed association with molecular subtypes of breast cancer namely luminal versus non luminal cases that may serve as an excellent marker for clinical decision making.

Budding uninhibited by benzimidazoles 1 promotes cell proliferation, invasion, and epithelial-mesenchymal transition via the Wnt/β-catenin signaling in glioblastoma

The pathogenesis and progression of GBM (glioblastoma), as one of the most frequently occurring malignancies of the central nervous system, are regulated by several genes. BUB1 (budding uninhibited by benzimidazoles 1) is a mitotic checkpoint that plays an important role in chromosome segregation as well as in various tumors. However, its role in glioma is unknown. The current study discovered prominently elevated BUB1 in glioma and a significant relationship between BUB1 expression, a high World Health Organization grade, and a poor prognosis in glioma patients. Moreover, BUB1 triggered EMT (epithelial-mesenchymal transition) apart from promoting glioma cell proliferation, migration, and infiltration. Besides, BUB1 promoted EMT by activating the Wnt/β-catenin axis. As implied by our study, BUB1 probably has the potential as a target for GBM management.

Moonlighting at the Poles: Non-Canonical Functions of Centrosomes.

Centrosomes are best known as the microtubule organizing centers (MTOCs) of eukaryotic cells. In addition to their classic role in chromosome segregation, centrosomes play diverse roles unrelated to their MTOC activity during cell proliferation and quiescence. Metazoan centrosomes and their functional doppelgängers from lower eukaryotes, the spindle pole bodies (SPBs), act as important structural platforms that orchestrate signaling events essential for cell cycle progression, cellular responses to DNA damage, sensory reception and cell homeostasis. Here, we provide a critical overview of the unconventional and often overlooked roles of centrosomes/SPBs in the life cycle of eukaryotic cells.

Mediator recruits the cohesin loader Scc2 to RNA Pol II-transcribed genes and promotes sister chromatid cohesion

SummaryThe ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 functions as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate the localization of the Scc2-Scc4 cohesin loader. Here, we identify a broad range of Scc2-chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in the recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and the decreased binding of Scc2 at RNA Pol II-transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in the direct recruitment of Scc2 to RNA Pol II-transcribed genes.

Combination of CENP-B Box Positive and Negative Synthetic Alpha Satellite Repeats Improves De Novo Human Artificial Chromosome Formation.

Human artificial chromosomes (HACs) can be formed de novo by introducing large (>30 kb) centromeric sequences consisting of highly repeated 171-bp alpha satellite (alphoid) DNA into HT1080 cells. However, only a subset of transformed cells successfully establishes HACs. CENP-A chromatin and heterochromatin assemble on the HACs and play crucial roles in chromosome segregation. The CENP-B protein, which binds a 17-bp motif (CENP-B box) in the alphoid DNA, functions in the formation of alternative CENP-A chromatin or heterochromatin states. A balance in the coordinated assembly of these chromatin states on the introduced alphoid DNA is important for HAC formation. To obtain information about the relationship between chromatin architecture and de novo HAC formation efficiency, we tested combinations of two 60-kb synthetic alphoid sequences containing either tetO or lacO plus a functional or mutated CENP-B box combined with a multiple fusion protein tethering system. The combination of mutated and wild-type CENP-B box alphoid repeats significantly enhanced HAC formation. Both CENP-A and HP1α were enriched in the wild-type alphoid DNA, whereas H3K27me3 was enriched on the mutant alphoid array. The presence or absence of CENP-B binding resulted in differences in the assembly of CENP-A chromatin on alphoid arrays and the formation of H3K9me3 or H3K27me3 heterochromatin.

The Interplay of Cohesin and RNA Processing Factors: The Impact of Their Alterations on Genome Stability

Cohesin, a multi-subunit protein complex, plays important roles in sister chromatid cohesion, DNA replication, chromatin organization, gene expression, transcription regulation, and the recombination or repair of DNA damage. Recently, several studies suggested that the functions of cohesin rely not only on cohesin-related protein–protein interactions, their post-translational modifications or specific DNA modifications, but that some RNA processing factors also play an important role in the regulation of cohesin functions. Therefore, the mutations and changes in the expression of cohesin subunits or alterations in the interactions between cohesin and RNA processing factors have been shown to have an impact on cohesion, the fidelity of chromosome segregation and, ultimately, on genome stability. In this review, we provide an overview of the cohesin complex and its role in chromosome segregation, highlight the causes and consequences of mutations and changes in the expression of cohesin subunits, and discuss the RNA processing factors that participate in the regulation of the processes involved in chromosome segregation. Overall, an understanding of the molecular determinants of the interplay between cohesin and RNA processing factors might help us to better understand the molecular mechanisms ensuring the integrity of the genome.

C9ORF78 partially localizes to centromeres and plays a role in chromosome segregation

C9ORF78 is a poorly characterized protein found in diverse eukaryotes. Previous work indicated overexpression of C9ORF78 in malignant tissues indicating a possible involvement in growth regulatory pathways. Additional studies in fission yeast and humans uncover a potential function in regulating the spliceosome. In studies of GFP-tagged C9ORF78 we observed a dramatic reduction in protein abundance in cells grown to confluence and/or deprived of serum growth factors. Serum stimulation induced synchronous re-expression of the protein in HeLa cells. This effect was also observed with the endogenous protein. Overexpressing either E2F1 or N-Myc resulted in elevated C9ORF78 expression potentially explaining the serum-dependent upregulation of the protein. Immunofluorescence analysis indicates that C9ORF78 localizes to nuclei in interphase but does not appear to concentrate in speckles as would be expected for a splicing protein. Surprisingly, a subpopulation of C9ORF78 co-localizes with ACA, Mad1 and Ndc80 in mitotic cells suggesting that this protein associates with kinetochores or centromeres. Levels of C9ORF78 at the centromere/kinetochore also increased upon activation of the mitotic checkpoint. Furthermore, knocking-down C9ORF78 caused mitotic defects. These studies uncover novel mitotic function and subcellular localization of C9ORF78.

Deficiency of nde1 in zebrafish induces brain inflammatory responses and autism-like behavior

SummaryThe cytoskeletal protein NDE1 plays an important role in chromosome segregation, neural precursor differentiation, and neuronal migration. Clinical studies have shown that NDE1 deficiency is associated with several neuropsychiatric disorders including autism. Here, we generated nde1 homologous deficiency zebrafish (nde1−/−) to elucidate the cellular molecular mechanisms behind it. nde1−/− exhibit increased neurological apoptotic responses at early infancy, enlarged ventricles, and shrank valvula cerebelli in adult brain tissue. Behavioral analysis revealed that nde1−/− displayed autism-like behavior traits such as increased locomotor activity and repetitive stereotype behaviors and impaired social and kin recognition behaviors. Furthermore, nde1 mRNA injection rescued apoptosis in early development, and minocycline treatment rescued impaired social behavior and overactive motor activity by inhibiting inflammatory cytokines. In this study, we revealed that nde1 homozygous deletion leads to abnormal neurological development with autism-related behavioral phenotypes and that inflammatory responses in the brain are an important molecular basis behind it.

Minichromosome maintenance proteins in eukaryotic chromosome segregation.

Minichromosome maintenance (Mcm) proteins are well-known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS-nonspecific group consisting of Mcm13, Mcm16-19, and Mcm21-22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high-fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non-overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure-function of the ARS-nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms.

Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression.

H2A.Z is a H2A‐type histone variant essential for many aspects of cell biology, ranging from gene expression to genome stability. From deuterostomes, H2A.Z evolved into two paralogues, H2A.Z.1 and H2A.Z.2, that differ by only three amino acids and are encoded by different genes (H2AFZ and H2AFV, respectively). Despite the importance of this histone variant in development and cellular homeostasis, very little is known about the individual functions of each paralogue in mammals. Here, we have investigated the distinct roles of the two paralogues in cell cycle regulation and unveiled non‐redundant functions for H2A.Z.1 and H2A.Z.2 in cell division. Our findings show that H2A.Z.1 regulates the expression of cell cycle genes such as Myc and Ki‐67 and its depletion leads to a G1 arrest and cellular senescence. On the contrary, H2A.Z.2, in a transcription‐independent manner, is essential for centromere integrity and sister chromatid cohesion regulation, thus playing a key role in chromosome segregation.

Novel Roles of Separase in Hematopoiesis

Separase, an enzyme that resolves sister chromatid cohesion during the metaphase-to-anaphase transition, plays a pivotal role in chromosomal segregation and cell division. In recent years, cancer genome sequencing has revealed several loss of function cohesin mutations in early stages of myeloid, hematologic and epithelial malignancies. These mutations affect the self-renewal and differentiation capacity of HSCs indicating cohesin as a tumor suppressor. Our prior studies indicated that Separase haploinsufficiency acts synergistically with loss of p53 in the initiation and progression of B- and T- cell lymphomas. In contrast to cohesin proteins, the function of the cohesin protease, Separase in hematopoiesis and hematological malignancies has not been well defined.Here we examined the effect of Espl1 heterozygosity using our hypomorphic mouse model (mEspl1 +/hyp ) that has reduced germline Separase activity. The impact of heterozygous loss of mEspl1 allele on total number of HSCs was analyzed by staining with hematopoietic stem and progenitor cells (HSPCs) markers and FACS analysis of cells isolated from bone marrow. Our analysis showed that Separase mutant mice have significantly increased (~2 fold) immunophenotypic HSPCs compared to wild type littermates. To examine whether reduced Separase level elevates self-renewal capacity of HSCs in vivo , we performed competitive transplantation assays to compare differentiation and self-renewal capacity of HSCs in mEspl1+/hyp and WT littermate controls. The outcomes with significantly higher donor derived reconstitution suggested an expansion of HSPCs compartment in mutant mice. In addition, we also observed an altered hematopoietic differentiation with an increase in percentage of donor-derived T- and B-cells in Separase mutant mice. The increased number of HSCs could be a consequence of change in autonomous function of the cells due to reduced level of Separase. To test the cell autonomous function of mEspl1+/hyp , in vitro colony forming unit (CFU) assays were performed in methylocellulose medium. mEspl1+/hyp cells showed significantly higher serial re-plating capacity compared to WT littermate control cells, supporting the notion of cell autonomous function as a probable cause of the observed phenotype.Considering the role of cohesin on gene expression, the effect of heterozygous loss of Separase was analyzed with global transcriptional profiling. Several hematopoietic fingerprint genes like Tle1, Smarca1, Scl2a10, H19, Krt2-8 and Galnt3 were found upregulated. Other genes important in HSC function such as Runx1, Satb1, Hoxa9 and Pbx1 were also upregulated in mEspl1+/hyp mice. The transcriptional profiling identified genes involved in several biological processes such as leukocyte differentiation, cell apoptosis, and chromatin modifications. However, mechanism and direct gene targets of Separase in hematopoiesis or other biological pathways are yet to be identified. To study further on transcriptional regulatory roles and to characterize the direct targets of Separase, ChIP-Seq (cohesin and Separase) and RNA-Seq experiments are being performed in WT and mEspl1+/hyp HSCs.With these studies we hope to better understand the role of cohesin in association with Separase on hematopoiesis particularly the mechanisms that control the fate of HSCs, bone marrow engraftment and function; how cohesin mutations may give rise to malignant transformation, and if this pathway can be targeted for therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Understanding The Role of Bir1 Sumoylation in Mitotic Regulation

To ensure the integrity of genome over generations, organisms have evolved multiple mechanisms to respond to DNA damage or chromosome instability. Protein sumoylation is one such mechanism for genome maintenance. Sumoylation describes a process in which concerted action of E1, E2 and E3 ligases facilitates the covalent linkage of small ubiquitin-like modifiers (SUMO) peptide to target proteins. In Saccharomyces cerevisiae, Siz1, Siz2 and Mms21 are three E3 SUMO ligases. Each of these ligases has preferred targets and it is generally accepted that they contribute to genome stability. Understanding specific SUMO targets will reveal mechanistic insights into the role of sumoylation in genome maintenance. In S. cerevisiae, Bir1, a subunit of chromosomal passenger complex (CPC), is a target of Mms21. During mitosis, CPC, a four-subunit complex composed of Bir1, Ipl1, Sli15 and Nbl1, mediates the spindle assembly checkpoint (SAC) to establish chromosome bi-orientation. Appropriate tension between two sister chromatids serves as a molecular signal for successful bi-orientation. Lack of tension may be a sign of incorrect attachment of mitotic spindles to the chromosome or unresolved replication intermediates resulting from DNA damage accumulation. Bir1, along with the CPC complex, plays a pivotal role in this process and the goal of our research is to understand the functional significance of Bir1 sumoylation. We hypothesize that sumoylation contributes to Bir1's regulation of mitotic progression. An existing piece of evidence supports this model: a decrease in sumoylation of Bir1 or Sli15 correlates with SAC deactivation when cells were treated with a genotoxic agent, methyl methanesulfonate (MMS). We genetically modified BIR1 to mimic conditions in which Bir1 is constantly sumoylated (BIR1-SuON) or cannot be sumoylated (BIR1-SuOff). These genetically modified strains are not sensitive to DNA damaging agents such as MMS or ultraviolet radiation similar to previously reported temperature sensitive mutants of Bir1 (bir1-107, bir1-110 and bir1-111) or Ipl1 (ipl1-321). This observation is different from the phenotype of mms21 mutants, which are known to be sensitive to genotoxic agents, such as MMS. These data together suggest that sumoylation of Bir1 by Mms21 is not responsible for resisting cell death when genome stability is challenged. Consistent with this idea, we observed that BIR1-SuOn allele did not rescue mms21 mutants’ sensitivity to MMS treatment. Intriguingly, we observed that both ipl1-321 and BIR1-SuOff mutants grew better than the control when cells were treated with high concentration of MMS. Similarly, it has been reported that hydroxyurea or benomylcan suppress ipl1-321 mutants’ temperature sensitivity. These observations suggest two possibilities: 1) sumoylated Bir1 shares some functions with Ipl1 in regulating chromosome segregation or 2) both BIR1-SuOff and ipl1-321 mutants have relatively unstable genome that allows them to generate revertants in the presence of genotoxic agents. To further test the idea that sumoylated Bir1 has a role in chromosome segregation, we are currently studying the phenotype of bir1 SUMO mutants in genetic background that disrupts mitotic regulation.

Ubiquitination in Plant Meiosis: Recent Advances and High Throughput Methods.

Meiosis is a specialized cell division which is essential to sexual reproduction. The success of this highly ordered process involves the timely activation, interaction, movement, and removal of many proteins. Ubiquitination is an extraordinarily diverse post-translational modification with a regulatory role in almost all cellular processes. During meiosis, ubiquitin localizes to chromatin and the expression of genes related to ubiquitination appears to be enhanced. This may be due to extensive protein turnover mediated by proteasomal degradation. However, degradation is not the only substrate fate conferred by ubiquitination which may also mediate, for example, the activation of key transcription factors. In plant meiosis, the specific roles of several components of the ubiquitination cascade—particularly SCF complex proteins, the APC/C, and HEI10—have been partially characterized indicating diverse roles in chromosome segregation, recombination, and synapsis. Nonetheless, these components remain comparatively poorly understood to their counterparts in other processes and in other eukaryotes. In this review, we present an overview of our understanding of the role of ubiquitination in plant meiosis, highlighting recent advances, remaining challenges, and high throughput methods which may be used to overcome them.