The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development

The molecular mechanisms that regulate cell cycle progression in a developmental context are poorly understood. Here, we show that the leucine-rich repeat protein LRR-1 promotes cell cycle progression during C. elegans development, both in the germ line and in the early embryo. Our results indicate that LRR-1 acts as a nuclear substrate-recognition subunit of a Cullin 2-RING E3 ligase complex (CRL2(LRR-1)), which ensures DNA replication integrity. LRR-1 contains a typical BC/Cul-2 box and binds CRL2 components in vitro and in vivo in a BC/Cul-2 box-dependent manner. Loss of lrr-1 function causes cell cycle arrest in the mitotic region of the germ line, resulting in sterility due to the depletion of germ cells. Inactivation of the DNA replication checkpoint signaling components ATL-1 and CHK-1 suppresses this cell cycle arrest and, remarkably, restores lrr-1 mutant fertility. Likewise, in the early embryo, loss of lrr-1 function induces CHK-1 phosphorylation and a severe cell cycle delay in P lineage division, causing embryonic lethality. Checkpoint activation is not constitutive in lrr-1 mutants but is induced by DNA damage, which may arise due to re-replication of some regions of the genome as evidenced by the accumulation of single-stranded DNA-replication protein A (ssDNA-RPA-1) nuclear foci and the increase in germ cell ploidy in lrr-1 and lrr-1; atl-1 double mutants, respectively. Collectively, these observations highlight a crucial function of the CRL2(LRR-1) complex in genome stability via maintenance of DNA replication integrity during C. elegans development.     

驱动蛋白Kin11调控嗜热四膜虫生殖系小核的减数分裂

驱动蛋白(kinesin)是分子马达蛋白质超家族成员,主要参与囊泡与细胞器的运输、纺锤体组装、有丝分裂和减数分裂等过程。在减数分裂期,不同驱动蛋白发挥功能的调控机制并不十分清楚。嗜热四膜虫(Tetrahymena thermophila)中含有14个驱动蛋白家族成员。其中,kinesin-6家族的唯一成员Kin11(TTHERM_00637750),在营养生长期低表达,饥饿期不表达,有性生殖期表达上调。Kin11编码1608个氨基酸,包含1个N端保守的马达蛋白结构域,C端卷曲螺旋(coiled-coil)结构域,并在N端和C端分别含有核定位信号NLS1和NLS2。Kin11在营养生长期和有性生殖期,定位在有丝分裂和减数分裂的小核和纺锤体上,并在有性生殖后期alignment阶段定位于小核上。Kin11与微管蛋白共定位于有丝分裂和减数分裂的纺锤体上。将Kin11的N端含有NLS1的1~400位氨基酸序列截短后,截断突变体定位在有性生殖减数分裂期的小核和纺锤体上。而将其C端含有NLS2的1008~1608位氨基酸残基截短后,截断突变体只能定位在有丝分裂和减数分裂后期的小核及有丝分裂的纺锤体上。敲除KIN11导致减数分裂过程中的纺锤体结构发生异常变化,小核染色体不均等分离与丢失,有性生殖发育停滞。结果表明,嗜热四膜虫驱动蛋白Kin11通过影响纺锤体结构,参与调控四膜虫生殖系小核在减数分裂过程中的正常分离。     

npr-1 Regulates foraging and dispersal strategies in Caenorhabditis elegans

Wild isolates of Caenorhabditis elegans differ in their tendency to aggregate on food [1, 2]. Most quantitative variation in this behavior is explained by a polymorphism at a single amino acid in the G protein-coupled receptor NPR-1: gregarious strains carry the 215F allele, and solitary strains carry the 215V allele [2]. Although npr-1 regulates a behavioral syndrome with potential adaptive implications, the evolutionary causes and consequences of this natural polymorphism remain unclear. Here we show that npr-1 regulates two behaviors that can promote coexistence of the two alleles. First, gregarious and solitary worms differ in their responses to food such that they can partition a single, continuous patch of food. Second, gregarious worms disperse more readily from patch to patch than do solitary worms, which can cause partitioning of a fragmented resource. The dispersal propensity of both gregarious and solitary worms increases with density. npr-1-dependent dispersal is independent of aggregation and could be part of a food-searching strategy. The gregarious allele is favored in a fragmented relative to a continuous food environment in competition experiments. We conclude that the npr-1 polymorphism could be maintained by a trade-off between dispersal and competitive ability.     

The C. elegans Homolog of RBBP6 (RBPL-1) Regulates Fertility through Controlling Cell Proliferation in the Germline and Nutrient Synthesis in the Intestine

RBBP6 (retinoblastoma binding protein 6, also known as PACT or P2P-R in humans) is a multi-domain protein that functions in multiple processes, such as mitosis, cell differentiation, and cell apoptosis. RBBP6 is evolutionarily conserved and is present in unicellular organisms to mammals. Studies of RBBP6 have mostly focused on its RB- and p53-binding domains, which are found exclusively in mammals. Here, we investigated the C. elegans homolog of RBBP6 to explore the functional roles of its other domains. We found that RBPL-1, the homolog of RBBP6 in C. elegans, is indispensable for worm development. RNAi silencing of rbpl-1 led to embryonic lethality, as well as defects in oocyte production and intestine development. rbpl-1 RNAi worms showed defects in germ cell proliferation, suggesting that RBPL-1 regulates mitosis. Moreover, RNAi silencing of rbpl-1 inhibited nutrient synthesis in the worm intestine. RBPL-1, as a nucleolus protein, was found to be expressed in diverse tissues and necessary for both germline and soma development. Using microarray analysis, we identified ≈700 genes whose expression levels were changed at least 10-fold in rbpl-1 worms. We propose that RBPL-1, like its yeast homolog, may regulate gene expression as an mRNA cleavage and polyadenylation factor. Taken together, the findings from this study reveal that RBPL-1 plays a pivotal role in C. elegans germline and soma development, suggesting that the functions of RBBP6 are conserved in diverse eukaryotic species.     

Germline Expression Influences Operon Organization in the Caenorhabditis elegans Genome

Operons are found across multiple kingdoms and phyla, from prokaryotes to chordates. In the nematode Caenorhabditis elegans, the genome contains >1000 operons that compose ~15% of the protein-coding genes. However, determination of the force(s) promoting the origin and maintenance of operons in C. elegans has proved elusive. Compared to bacterial operons, genes within a C. elegans operon often show poor coexpression and only sometimes encode proteins with related functions. Using analysis of microarray and large-scale in situ hybridization data, we demonstrate that almost all operon-encoded genes are expressed in germline tissue. However, genes expressed during spermatogenesis are excluded from operons. Operons group together along chromosomes in local clusters that also contain monocistronic germline-expressed genes. Additionally, germline expression of genes in operons is largely independent of the molecular function of the encoded proteins. These analyses demonstrate that mechanisms governing germline gene expression influence operon origination and/or maintenance. Thus, gene expression in a specific tissue can have profound effects on the evolution of genome organization.     

CRL2(LRR-1) targets a CDK inhibitor for cell cycle control in C. elegans and actin-based motility regulation in human cells

The Cip/Kip CDK inhibitor (CKI) p21(Cip1/WAF1) has?a critical role in the nucleus to limit cell proliferation by inhibiting CDK-cyclin complexes. In contrast, cytoplasmic p21 regulates cell survival and the actin cytoskeleton. These divergent functions for p21 in different cellular compartments suggest the necessity for complex regulation. In this study, we identify the CRL2(LRR-1) ubiquitin ligase as a conserved regulator of Cip/Kip CKIs that promotes the degradation of C. elegans CKI-1 and human p21. The nematode CRL2(LRR-1) complex negatively regulates nuclear CKI-1 levels to ensure G1-phase cell cycle progression in germ cells. In contrast, human CRL2(LRR1) targets cytoplasmic p21, acting as a critical regulator of cell motility that promotes a nonmotile stationary cell state by preventing p21 from inhibiting the Rho/ROCK/LIMK pathway. Inactivation of human CRL2(LRR1) leads to the activation of the actin-depolymerizing protein cofilin, dramatic reorganization of the actin cytoskeleton, and increased cell motility.     

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.     

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.     

SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4-dependent endonuclease activity toward replication forks, 5'-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.     

SATB1 Overexpression Regulates the Development and Progression in Bladder Cancer through EMT

The global gene regulator Special AT-rich sequence-binding protein-1 (SATB1) has been reported to induce EMT-like changes and be associated with poor clinical outcome in several cancers. This study aims to evaluate whether SATB1 affects the biological behaviors of bladder transitional cell carcinoma (BTCC) and further elucidate if this effect works through an epithelial-mesenchymal transition (EMT) pathway. The expression of SATB1, E-cadherin (epithelial markers), vimentin (mesenchymal markers) in BTCC tissues and adjacent noncancerous tissues, as well as in two cell lines of bladder cancer were investigated. Whether the SATB1 expression is associated with clinicopathological factors or not was statistically analyzed. Cell invasion and migration, cell cycle, cell proliferation and apoptosis were evaluated in SATB1 knockdown and overexpressed cell lines. Our results showed that the expression of SATB1 was remarkably up-regulated both in BTCC tissues and in bladder cancer cell lines with high potential of metastasis. The results were also associated with EMT markers and poor prognosis of BTCC patients. Moreover, SATB1 induced EMT processes through downregulation of E-cadherin, upregulation of E-cadherin repressors (Snail, Slug and vimentin). SATB1 also promoted cell cycle progression, cell proliferation, cell invasion and cell migration, but did not alter cell survival. In conclusion, our results suggest that SATB1 plays a crucial role in the progression of bladder cancer by regulating genes controlling EMT processes. Further, it may be a novel therapeutic target for aggressive bladder cancers.     

CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation

The human inhibitor of Bruton''s tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3^IBTK complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3^IBTK for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3^IBTK regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5′-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3^IBTK as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.     

Joint Molecule Resolution Requires the Redundant Activities of MUS-81 and XPF-1 during Caenorhabditis elegans Meiosis

The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in Caenorhabditis elegans, SPO-11-generated double stranded breaks are resolved to generate a single crossover per bivalent and the remaining recombination intermediates are resolved as noncrossovers. We discovered that early recombination intermediates are limited by the C. elegans BLM ortholog, HIM-6, and in the absence of HIM-6 by the structure specific endonuclease MUS-81. In the absence of both MUS-81 and HIM-6, recombination intermediates persist, leading to chromosome breakage at diakinesis and inviable embryos. MUS-81 has an additional role in resolving late recombination intermediates in C. elegans. mus-81 mutants exhibited reduced crossover recombination frequencies suggesting that MUS-81 is required to generate a subset of meiotic crossovers. Similarly, the Mus81-related endonuclease XPF-1 is also required for a subset of meiotic crossovers. Although C. elegans gen-1 mutants have no detectable meiotic defect either alone or in combination with him-6, mus-81 or xpf-1 mutations, mus-81;xpf-1 double mutants are synthetic lethal. While mus-81;xpf-1 double mutants are proficient for the processing of early recombination intermediates, they exhibit defects in the post-pachytene chromosome reorganization and the asymmetric disassembly of the synaptonemal complex, presumably triggered by crossovers or crossover precursors. Consistent with a defect in resolving late recombination intermediates, mus-81; xpf-1 diakinetic bivalents are aberrant with fine DNA bridges visible between two distinct DAPI staining bodies. We were able to suppress the aberrant bivalent phenotype by microinjection of activated human GEN1 protein, which can cleave Holliday junctions, suggesting that the DNA bridges in mus-81; xpf-1 diakinetic oocytes are unresolved Holliday junctions. We propose that the MUS-81 and XPF-1 endonucleases act redundantly to process late recombination intermediates to form crossovers during C. elegans meiosis.