Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.     

Nicotiana attenuata SIPK,WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.Jasmonic acid (JA) and some of its precursors and derivatives are signal molecules that function as essential mediators of the plant''s wound, antiherbivore, and antipathogen responses, as well as in growth and development (Farmer, 1994; Creelman and Mullet, 1997; Turner et al., 2002). In unelicited mature leaves, JA is maintained at very low levels, however, upon specific stimulations, its biosynthesis is induced within a few minutes (Glauser et al., 2008). This rapid biosynthetic response must result from the activation of constitutively expressed JA biosynthesis enzymes in unelicited tissue by substrate availability and/or posttranslational modifications. At present, little is known about the molecular mechanisms that activate JA biosynthetic enzymes.According to the canonical mechanism for JA biosynthesis (Vick and Zimmerman, 1983), free α-linolenic acid (18:3^Δ9,12,15, 18:3) forms 13(S)-hydroperoxyoctadecatrienoic acid [13S-(OOH)-18:3] by the action of 13-lipoxygenase (13-LOX) in plastids. 13S-(OOH)-18:3 is converted by allene oxide synthase (AOS) into a highly unstable allene oxide intermediate that is processed by allene oxide cyclase (AOC) to yield (9S,13S)-12-oxo-phytodienoic acid (OPDA). OPDA is transported from the plastid into the peroxisome where it is reduced by the action of OPDA reductase 3 (OPR3) and after three cycles of β-oxidation, (3R,7S)-JA is formed. Due to the large number of enzymes and different cellular compartments involved in JA biosynthesis, it is expected that the pathway is regulated at multiple steps. Resolution of the structures of the tomato (Solanum lycopersicum) OPR3 and Arabidopsis (Arabidopsis thaliana) AOC2 and ACX1 has provided insights into potential regulatory mechanisms for these enzymes (e.g. oligomerization and phosphorylation; Pedersen and Henriksen, 2005; Breithaupt et al., 2006; Hofmann et al., 2006).The identification of two Arabidopsis plastidial glycerolipases, DAD1 and DGL (Ishiguro et al., 2001; Hyun et al., 2008), has provided genetic evidence for the importance of the release of trienoic fatty acids (FAs) from plastidial lipids in the activation of JA biosynthesis. Recently, some oxylipins have been found esterified to galactolipids in Arabidopsis leaves and hence it is possible that in this species preformed precursors could also supply the JA biosynthesis pathway after their release from lipids (Stelmach et al., 2001; Hisamatsu et al., 2003; Buseman et al., 2006). However, lipid-bound oxylipins are not formed in the leaves of all plant families (Böttcher and Weiler, 2007).In Nicotiana attenuata, wound-induced JA production is amplified by the application of lepidopteran larvae (e.g. Manduca sexta) oral secretions (OS) to mechanical wounds. Major elicitors of the OS-mediated response are FA-amino acid conjugates (FACs) that are sufficient to enhance JA production in leaves of this plant species (Halitschke et al., 2001). Recently, several regulatory factors with a potential function upstream of JA biosynthesis have been identified (Ludwig et al., 2005; Takabatake et al., 2006; Schweighofer et al., 2007; Takahashi et al., 2007); however, how these regulators affect JA biosynthesis is at present unknown. For example, wounding and herbivory in Nicotina spp. and tomato activate the mitogen-activated protein kinases salicylate-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK; Seo et al., 1999; Kandoth et al., 2007; Wu et al., 2007). When SIPK and WIPK expression is silenced in tobacco (Nicotiana tabacum), the plants accumulate 60% to 70% less JA than wild type after wounding or OS elicitation (Seo et al., 2007; Wu et al., 2007). Another regulatory component that affects JA production in N. attenuata is Nonexpressor of PR-1 (NPR1), an essential component of the salicylic acid (SA) signal transduction pathway first identified in Arabidopsis (Cao et al., 1994). N. attenuata NPR1-silenced plants accumulate 60% to 70% lower JA levels after elicitation than wild type (Rayapuram and Baldwin, 2007). NPR1 interacts with the JA and ethylene signaling cascades, and a cytosolic role for this factor in the regulation of JA-dependent responses/biosynthesis has been proposed (Spoel et al., 2003).In contrast to the mechanisms acting upstream of JA biosynthesis, the mechanisms mediating downstream JA responses are better characterized (Kazan and Manners, 2008; Browse, 2009). Among the best-characterized regulators of these responses is CORONATIVE INSENSITIVE1 (COI1), a gene that participates in jasmonate perception (Xie et al., 1998) and regulates gene expression through its interaction with the JASMONATE ZIM-DOMAIN repressors (Chini et al., 2007; Thines et al., 2007).To understand the early processes regulating the activation of JA biosynthesis by wounding and FAC elicitation in N. attenuata leaves, we quantified the initial rates of accumulation of plastid-derived JA precursors after these stimuli in wild type and four JA-deficient genotypes previously described: ir-sipk, ir-wipk, ir-npr1, and ir-coi1 (Rayapuram and Baldwin, 2007; Paschold et al., 2008; Meldau et al., 2009). We show that SIPK, WIPK, NPR1, and FACs contribute to the activation of de novo JA biosynthesis by affecting diverse early enzymatic steps in this pathway. The identification of a plastidial glycerolipase A₁ type I family protein (GLA1) essential for JA biosynthesis pointed to this enzyme as one potential target of some of these activating mechanisms.     

Egg trading in the simultaneously hermaphroditic polychaete worm Ophryotrocha gracilis (Huth)

Ophryotrocha gracilis (Huth 1934) is a simultaneously hermaphroditicpolychaete worm with external fertilization and a brief protandrousphase. The mating system of this species seems to meet conditionsleading to the establishment of egg-trading behavior. Experimentsshowed that mating occurs in pairs composed of two simultaneoushermaphrodites; sex roles are sequentially alternated and self-fertilizationis avoided. Egg reciprocation is kept evolutionarily stableby laying eggs in multiple, small-sized egg clutches and reducingthe reproductive success of pairs in which one of the two partnersdoes not reciprocate egg exchange. The frequency of ovigeroushermaphrodites in mass cultures of O. gracilis is about 50".Such a high mate availability preadapts hermaphrodites of O.gracilis to change partners very frequently and to reduce investmentin parental care, contrary to what is observed in another egg-trading,simultaneously hermaphroditic species, O. diadema. Laboratorypopulations of O. diadema have a frequency of only 17% ovigeroushermaphrodites     

AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody

To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.     

Mice with reduced expression of the telomere‐associated protein Ft1 develop p53‐sensitive progeroid traits

Human AKTIP and mouse Ft1 are orthologous ubiquitin E2 variant proteins involved in telomere maintenance and DNA replication. AKTIP also interacts with A‐ and B‐type lamins. These features suggest that Ft1 may be implicated in aging regulatory pathways. Here, we show that cells derived from hypomorph Ft1 mutant (Ft1^kof/kof) mice exhibit telomeric defects and that Ft1^kof/kof animals develop progeroid traits, including impaired growth, skeletal and skin defects, abnormal heart tissue, and sterility. We also demonstrate a genetic interaction between Ft1 and p53. The analysis of mice carrying mutations in both Ft1 and p53 (Ft1^kof/kof; p53^ko/ko and Ft1^kof/kof; p53^+/ko) showed that reduction in p53 rescues the progeroid traits of Ft1 mutants, suggesting that they are at least in part caused by a p53‐dependent DNA damage response. Conversely, Ft1 reduction alters lymphomagenesis in p53 mutant mice. These results identify Ft1 as a new player in the aging process and open the way to the analysis of its interactions with other progeria genes using the mouse model.     

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT‐stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral‐MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.