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Invadolysin Acts Genetically Via the SAGA Complex to Modulate Chromosome Structure Shubha Gururaja Rao†, Michal M

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Invadolysin Acts Genetically Via the SAGA Complex to Modulate Chromosome Structure Shubha Gururaja Rao†, Michal M 3546–3562 Nucleic Acids Research, 2015, Vol. 43, No. 7 Published online 16 March 2015 doi: 10.1093/nar/gkv211 Invadolysin acts genetically via the SAGA complex to modulate chromosome structure Shubha Gururaja Rao†, Michal M. Janiszewski†, Edward Duca, Bryce Nelson, Kanishk Abhinav, Ioanna Panagakou, Sharron Vass and Margarete M.S. Heck* University of Edinburgh, Queen’s Medical Research Institute, University/BHF Centre for Cardiovascular Science, 47 Little France Crescent, Edinburgh EH16 4TJ, UK Received December 13, 2012; Revised February 26, 2015; Accepted February 28, 2015 Downloaded from https://academic.oup.com/nar/article/43/7/3546/2414574 by guest on 04 October 2021 ABSTRACT (gcn5, ada2b and sgf11) all suppress an invadolysin- induced rough eye phenotype. We conclude that Identification of components essential to chromo- the abnormal chromosome phenotype of invadolysin some structure and behaviour remains a vibrant mutants is likely the result of disrupting the histone area of study. We have previously shown that in- modification cycle, as accumulation of ubH2B and vadolysin is essential in Drosophila, with roles in cell H3K4me3 is observed. We further suggest that the division and cell migration. Mitotic chromosomes mislocalization of ubH2B to the cytoplasm has ad- are hypercondensed in length, but display an aber- ditional consequences on downstream components rant fuzzy appearance. We additionally demonstrated essential for chromosome behaviour. We therefore that in human cells, invadolysin is localized on the propose that invadolysin plays a crucial role in chro- surface of lipid droplets, organelles that store not mosome organization via its interaction with the only triglycerides and sterols but also free histones SAGA complex. H2A, H2Av and H2B. Is there a link between the storage of histones in lipid droplets and the aber- rantly structured chromosomes of invadolysin mu- INTRODUCTION tants? We have identified a genetic interaction be- The visible changes that chromosomes undergo through the tween invadolysin and nonstop, the de-ubiquitinating cell cycle have been the subject of intense cytological anal- protease component of the SAGA (Spt-Ada-Gcn5- ysis for over a century. Recently, many powerful techniques acetyltransferase) chromatin-remodelling complex. have been brought to bear on the subject of the reversible cy- invadolysin and nonstop mutants exhibit pheno- cle of chromosome condensation and decondensation. Bio- typic similarities in terms of chromosome struc- chemical, physical, genetic and cell biological approaches ture in both diploid and polyploid cells. Further- have all served to identify a myriad of structural and regu- more, IX-141/not1 transheterozygous animals accu- latory components whose detailed roles are still being elu- cidated (1). mulate mono-ubiquitinated histone H2B (ubH2B) and Chromatin condensation is particularly evident during histone H3 tri-methylated at lysine 4 (H3K4me3). mitosis and apoptosis, whereas chromatin relaxation is as- 1/ 1 Whole mount immunostaining of IX-14 not tran- sociated with transcription, replication, repair and recombi- sheterozygous salivary glands revealed that ubH2B nation (2). Post-translational modifications of histones, e.g. accumulates surprisingly in the cytoplasm, rather acetylation, methylation, phosphorylation and ubiquitina- than the nucleus. Over-expression of the Bre1 ubiq- tion, play key roles in chromatin dynamics (3,4). Acetyla- uitin ligase phenocopies the effects of mutating tion of histones is generally associated with ‘relaxing’ or either the invadolysin or nonstop genes. Intrigu- opening chromatin in order to support the access of tran- ingly, nonstop and mutants of other SAGA subunits scriptional machinery to genomic loci (5). In contrast, hi- stone methylation and ubiquitination have been linked to *To whom correspondence should be addressed. Email: [email protected] †These authors contributed equally to the paper as first authors. Present addresses: Shubha Gururaja Rao, Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA 19102, USA. Michal M. Janiszewski, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30–387, Krakow 31-007 Krakow, Poland. Edward Duca, University of Malta, Msida, MSD 2080, Malta. Bryce Nelson, Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA. Sharron Vass, Edinburgh Napier University, Edinburgh EH11 4BN, UK. C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2015, Vol. 43, No. 7 3547 both gene activation and repression (6). Specific phospho- MATERIALS AND METHODS rylation signatures have been identified for both mitosis Maintenance of Drosophila stocks and apoptosis. The enzymes that catalyse histone modifica- tion are often organized into large multi-subunit complexes, All fly stocks were maintained at 25˚C on standard medium which facilitate enzymatic activity and enhance substrate unless otherwise stated. The Canton S strain served as specificity. the wild type stock, and is indicated as ‘wt’ through the We previously reported the identification of invadolysin, manuscript. The deficiency collection was from DrosDel predicted to be a metalloprotease, which links the pro- (Szeged) and Gal4 driver flies were from the Bloomington cesses of mitosis and cell migration (7). invadolysin mu- Stock Centre. Nonstop mutant flies were a kind gift from I. tant larvae display defects in mitotic chromosome packag- Salecker and B. Poeck. UAS-Bre1 flies were a generous gift ing, appearing hypercondensed in length but with a ‘fuzzy’ from M. Bienz and S. Bray. Genetic crosses were performed or ragged appearance at the periphery. The chromosome using virgin female flies collected as soon as possible after phenotype was distinct from other chromosome conden- eclosion. Downloaded from https://academic.oup.com/nar/article/43/7/3546/2414574 by guest on 04 October 2021 sation mutations we analysed (8–10), and invadolysin mu- tant neuroblasts also exhibited defects in spindle assem- bly and nuclear envelope dynamics. The active migration of primordial germ cells was also affected in invadolysin Second site non-complementation screen mutant fly embryos, this being consistent with the localiza- The DrosDel deficiency kit was used as these deficiencies tion of invadolysin at the leading edge of migrating human have molecularly defined breakpoints and are originally de- macrophages (7). The human invadolysin protein is local- rived from an isogenic stock. Five to ten virgin females from ized to intracellular ring-like structures, which we subse- IX-141 mutant stocks over TM3 or TM6B were crossed with quently identified as lipid droplets11 ( ). A functional role two to three males from each of the 225 DrosDel deficiency for invadolysin in lipid metabolism is suggested by a de- lines. Flies were left to mate and lay eggs for 5 days at room creased triglyceride to protein ratio in mutant larvae (11). temperature. The parents were removed from the vials and While the gene is conserved from bacteria to higher eukary- the progeny were allowed to emerge in the same vial. These otes, it is curiously absent from archaea, fungi, and viruses. crosses were carried out at room temperature and after 12 There are four splice variants of human invadolysin, though days, the eclosed flies were scored for the presence or ab- so far only one has been described in Drosophila (11). sence of balancer chromosomes. Males and females were Proteases are crucial for homeostasis through the regu- counted separately in order to investigate if there were any lation of inter-protein and inter-cellular interactions. Met- gender specific phenotypes. alloproteases are a class of proteases that require either zinc or calcium for their activity (12). Metalloproteases from the MA clan contain a short consensus HEXXH sequence––the two histidines act as ligands for the zinc ion, Preparation of larval samples for SDS-PAGE while glutamate acts as a catalytic base (13). The M8 met- zincin family (to which invadolysin belongs) also encodes Five to thirty wandering third instar larvae of the appropri- a more C-terminal histidine that is a third ligand for zinc. ate genotype were removed from culture bottles and trans- Metalloproteases are involved in crucial processes affecting ferred to 1.5 ml Eppendorf tubes and rinsed 3× in EBR cell adhesion, cell migration, tissue remodelling and differ- (Ephrussi-Beadle Ringer’s solution: 130 mM NaCl, 4.7 mM entiation (14). Metalloproteases have been implicated in nu- KCl, 1.9 mM CaCl2, 10 mM HEPES, pH 6.9). One hun- merous regulatory roles emphasizing their diverse functions dred fifty microlitres of cold lysis buffer (EBR, 10mM and biological importance. ethylenediaminetetraacetic acid (EDTA), 10 mM dithio- In an attempt to better understand invadolysin, we de- threitol (DTT), 10 ␮g/ml each of chymostatin, leupeptin, scribe herein a second site non-complementation screen antipain and pepstatin [CLAP], 1 mM phenylmethanesul- performed in Drosophila to identify genes interacting with fonyl fluoride [PMSF], and 1 unit aprotinin) was added to invadolysin. We identified the gene responsible for
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