TRF2/RAP1 and DNA–PK mediate a double protection against joining at telomeric ends

DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA–PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA–PK end binding and activation step and (2) DNA–PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells.     

SNM1B/Apollo interacts with Astrin and is required for the prophase cell cycle checkpoint

Previously, we have shown that SNM1A is a multifunctional gene involved in both the DNA damage response and in an early mitotic checkpoint in response to spindle stress. Another member of the SNM1 gene family, SNM1B/Apollo, has been shown to have roles in both the response to DNA interstrand cross-linking agents and in telomere protection during S phase. Here, we demonstrate a novel role for SNM1B/Apollo in mitosis in response to spindle stress. SNM1B-deficient cells exhibit a defect in the prophase checkpoint. Loss of the prophase checkpoint induces an extended mitotic delay, which is due to prolonged activation of the spindle checkpoint. In addition, we show that SNM1B/Apollo interacts with the essential microtubule binding protein Astrin. SNM1B/Apollo interacts with Astrin through its conserved metallo-β-lactamase domain, and disruption of this interaction by point mutations results in a deficient prophase checkpoint. These findings suggest that SNM1B/Apollo and Astrin function together to enforce the prophase checkpoint in response to spindle stress.     

Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity

The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.     

Ribosomal protein S9 is a novel B23/NPM-binding protein required for normal cell proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.     

Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis‐segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.     

Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA) -mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.     

The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191(+/-) mice are normal and fertile. Homozygous Zfp191(-/-) embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191(-/-) and Zfp191(+/-) embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191(-/-) cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191(+/-) intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation.     

The dual-specificity protein phosphatase DUSP9/MKP-4 is essential for placental function but is not required for normal embryonic development

To elucidate the physiological role(s) of DUSP9 (dual-specificity phosphatase 9), also known as MKP-4 (mitogen-activated protein kinase [MAPK] phosphatase 4), the gene was deleted in mice. Crossing male chimeras with wild-type females resulted in heterozygous (DUSP9(+/-)) females. However, when these animals were crossed with wild-type (DUSP9(+/y)) males none of the progeny carried the targeted DUSP9 allele, indicating that both female heterozygous and male null (DUSP9(-/y)) animals die in utero. The DUSP9 gene is on the X chromosome, and this pattern of embryonic lethality is consistent with the selective inactivation of the paternal X chromosome in the extraembryonic tissues of the mouse, suggesting that DUSP9/MKP4 performs an essential function during placental development. Examination of embryos between 8 and 10.5 days postcoitum confirmed that lethality was due to a failure of labyrinth development, and this correlates exactly with the normal expression pattern of DUSP9/MKP-4 in the trophoblast giant cells and labyrinth of the placenta. Finally, when the placental defect was rescued, male null (DUSP9(-/y)) embryos developed to term, appeared normal, and were fertile. Our results indicate that DUSP9/MKP-4 is essential for placental organogenesis but is otherwise dispensable for mammalian embryonic development and highlights the critical role of dual-specificity MAPK phosphatases in the regulation of developmental outcomes in vertebrates.     

Microtubule-associated protein 1B (MAP1B) is required for dendritic spine development and synaptic maturation

Microtubule-associated protein 1B (MAP1B) is prominently expressed during early stages of neuronal development, and it has been implicated in axonal growth and guidance. MAP1B expression is also found in the adult brain in areas of significant synaptic plasticity. Here, we demonstrate that MAP1B is present in dendritic spines, and we describe a decrease in the density of mature dendritic spines in neurons of MAP1B-deficient mice that was accompanied by an increase in the number of immature filopodia-like protrusions. Although these neurons exhibited normal passive membrane properties and action potential firing, AMPA receptor-mediated synaptic currents were significantly diminished. Moreover, we observed a significant decrease in Rac1 activity and an increase in RhoA activity in the post-synaptic densities of adult MAP1B(+/-) mice when compared with wild type controls. MAP1B(+/-) fractions also exhibited a decrease in phosphorylated cofilin. Taken together, these results indicate a new and important role for MAP1B in the formation and maturation of dendritic spines, possibly through the regulation of the actin cytoskeleton. This activity of MAP1B could contribute to the regulation of synaptic activity and plasticity in the adult brain.     

Golga5 is dispensable for mouse embryonic development and postnatal survival

Golgins are a family of coiled‐coil proteins located at the cytoplasmic surface of the Golgi apparatus and have been implicated in maintaining Golgi structural integrity through acting as tethering factors for retrograde vesicle transport. Whereas knockdown of several individual golgins in cultured cells caused Golgi fragmentation and disruption of vesicle trafficking, analysis of mutant mouse models lacking individual golgins have discovered tissue‐specific developmental functions. Recently, homozygous loss of function of GOLGA2, of which previous in vitro studies suggested an essential role in maintenance of Golgi structure and in mitosis, has been associated with a neuromuscular disorder in human patients, which highlights the need for understanding the developmental roles of the golgins in vivo. We report here generation of Golga5‐deficient mice using CRISPR/Cas9‐mediated genome editing. Although knockdown studies in cultured cells have implicated Golga5 in maintenance of Golgi organization, we show that Golga5 is not required for mouse embryonic development, postnatal survival, or fertility. Moreover, whereas Golga5 is structurally closely related to Golgb1, we show that inactivation of Golga5 does not enhance the severity of developmental defects in Golgb1‐deficient mice. The Golga5‐deficient mice enable further investigation of the roles and functional specificity of golgins in development and diseases.     

Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly

The functions of Beclin‐1 in macroautophagy, tumorigenesis and cytokinesis are thought to be mediated by its association with the PI3K‐III complex. Here, we describe a new role for Beclin‐1 in mitotic chromosome congression that is independent of the PI3K‐III complex and its role in autophagy. Beclin‐1 depletion in HeLa cells leads to a significant reduction of the outer kinetochore proteins CENP‐E, CENP‐F and ZW10, and, consequently, the cells present severe problems in chromosome congression. Beclin‐1 associates with kinetochore microtubules and forms discrete foci near the kinetochores of attached chromosomes. We show that Beclin‐1 interacts directly with Zwint‐1—a component of the KMN (KNL‐1/Mis12/Ndc80) complex—which is essential for kinetochore–microtubule interactions. This suggests that Beclin‐1 acts downstream of the KMN complex to influence the recruitment of outer kinetochore proteins and promotes accurate kinetochore anchoring to the spindle during mitosis.     

NBS1 is required for IGF-1 induced cellular proliferation through the Ras/Raf/MEK/ERK cascade

NBS1 is a member of the Mre11–Rad50–NBS1 complex, which plays a role in cellular responses to DNA damage and the maintenance of genomic stability. Transgenic mice models and clinical symptoms of NBS patients have shown that NBS1 exerts pleiotropic actions on the growth and development of mammals. The present study showed that after repression of endogenous NBS1 levels using short interfering RNA, hTERT-RPE cells demonstrated impaired proliferation and a poor response to IGF-1. NBS1 down-regulated cells displayed disturbances in periodical oscillations of cyclin E and A and delayed cell cycle progression. Remarkably, lower phosphorylation levels of c-Raf and diminished activity of Erk1/2 in response to IGF-1 suggest a link among NBS1, IGF-1 signaling and the Ras/Raf/MEK/ERK cascade. The functional relevance of NBS1 in mitogenic signaling and initiation of cell cycle progression were demonstrated in NBS1 down-regulated cells where IGF-1 had a limited ability to induce the FOS and CCND1 expressions. In conclusion, our findings provide strong evidence that NBS1 has a functional role in IGF-1 signaling for the promotion of cell proliferation via the Ras/Raf/MEK/ERK cascade.     

poky/chuk/ikk1 is required for differentiation of the zebrafish embryonic epidermis

An epidermis surrounds all vertebrates, forming a water barrier between the external environment and the internal space of the organism. In the zebrafish, the embryonic epidermis consists of an outer enveloping layer (EVL) and an inner basal layer that have distinct embryonic origins. Differentiation of the EVL requires the maternal effect gene poky/ikk1 in EVL cells prior to establishment of the basal layer. This requirement is transient and maternal Ikk1 is sufficient to allow establishment of the EVL and formation of normal skin in adults. Similar to the requirement for Ikk1 in mouse epidermis, EVL cells in poky mutants fail to exit the cell cycle or express specific markers of differentiation. In spite of the similarity in phenotype, the molecular requirement for Ikk1 is different between mouse and zebrafish. Unlike the mouse, EVL differentiation requires functioning Poky/Ikk1 kinase activity but does not require the HLH domain. Previous work suggested that the EVL was a transient embryonic structure, and that maturation of the epidermis required replacement of the EVL with cells from the basal layer. We show here that the EVL is not lost during embryogenesis but persists to larval stages. Our results show that while the requirement for poky/ikk1 is conserved, the differences in molecular activity indicate that diversification of an epithelial differentiation program has allowed at least two developmental modes of establishing a multilayered epidermis in vertebrates.     

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.     

The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development

Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs. The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal cells.