Inaktiváló hatású fehérjekomplexek szerepe és működése a Drosophila homeotikus gének szabályozásában = Function of silencing complexes in the regulation of homeotic genes in Drosophila

Csoportunk a génkifejeződés epigenetikus szabályozását tanulmányozza a Drosophila bithorax génkomplexének, mint modellrendszernek a segítségével. Az elmúlt pályázati periódusban megkezdtük egy, a csoportunk által korábban létrehozott nagyméretű mutáns gyűjteménynek a feldolgozását, amely ismeretlen, Pc- és trx csoporba tartózó gének mutációit tartalmazza. Gyűjteményünk felhasználásával jellemeztük a grappa gént, ami az élesztő DOT1 fehérjéjének, egy, a H3-as hiszton K79-es pozícióját specifikusan metiláló enzim muslica homológját kódolja. Kimutattuk, hogy a korábban kizárólag transzkripciós aktivátornak gondolt GRAINYHEAD fehérje hozzájárul ahhoz, hogy a különböző PcG fehérjekomplexek specifikusan kötődjenek az iab-7 PRE-hez). Transgenikus vonalak felhasználásával térképeztünk egy trithorax response element-et (TRE) közvetlenül az iab-7 PRE szomszédságában. Kimutattuk, hogy az ENHANCER OF ZESTE (E(Z)) fehérje C-terminális darabja histon-metil-transzferáz aktivitást mutat, és hogy egy funkciónyeréses mutáns változat, az E(Z)TRM specificitása és hatékonysága csak kis mértékben különbözik a vad fehérje megfelelő tulajdonságaitól. Genetikai interakciós vizsgálataink felvetik azt a lehetőséget, hogy az E(Z) és különböző trxG fehérjék kompetálnak azokért a nukleoszómákért, amelyek közel vannak ahhoz a helyhez (PRE és TRE), ahová ezek e fehérjék specifikusan kötődnek. Jelentős előrehaladást értünk el több, általunk azonosított, Pc- vagy trx csoportba tartozó génnek a jellemzésében is. | Our group studies the epigenetic control of gene expression using the Drosophila bithorax complex as a model system. During the past granting period we begin to characterize a large mutant collection, containing new mutations belonging to either to the Pc-, or to the trx group of genes, that we generated earlier. Using this set of mutations we described and characterized the grappa gene that codes for the sole Drosophila homolog of the yeast DOT1 protein, a histone H3 K79 methyl-transferase. Also based on our mutations, we demonstrated that the GRAINYHEAD protein, which was originally thought to be a transcriptional activator, contributes to the specific targeting of PcG protein complexes to the infraabdominal-7 Polycomb response element, iab-PRE. Using transgenic lines, we mapped a trithorax response element (TRE) next to the iab-PRE. We showed that a truncated version of the PcG protein, ENHANCER OF ZESTE (E(Z)), possess histone-methyl-transferase activity, and that the specificity of a mutant, excess of function variant, E(Z)TRM, is only slightly different from the wild type. Genetic interaction studies suggested that E(Z) and different TRXG proteins may compete for common substrates, e. g. nucleosomes near to the site where these proteins are specifically targeted. We also made significant progress in characterizing several newly identified genes belonging either to the Pc-, or to the trx

Ion Channels and Pumps in Autophagy: A Reciprocal Relationship

Autophagy, the process of cellular self-degradation, is intrinsically tied to the degradative function of the lysosome. Several diseases have been linked to lysosomal degradative defects, including rare lysosomal storage disorders and neurodegenerative diseases. Ion channels and pumps play a major regulatory role in autophagy. Importantly, calcium signaling produced by TRPML1 (transient receptor potential cation channel, mucolipin subfamily) has been shown to regulate autophagic progression through biogenesis of autophagic-lysosomal organelles, activation of mTORC1 (mechanistic target of rapamycin complex 1) and degradation of autophagic cargo. ER calcium channels such as IP(3)Rs supply calcium for the lysosome, and lysosomal function is severely disrupted in the absence of lysosomal calcium replenishment by the ER. TRPML1 function is also regulated by LC3 (microtubule-associated protein light chain 3) and mTORC1, two critical components of the autophagic network. Here we provide an overview of the current knowledge about ion channels and pumps—including lysosomal V-ATPase (vacuolar proton-ATPase), which is required for acidification and hence proper enzymatic activity of lysosomal hydrolases—in the regulation of autophagy, and discuss how functional impairment of some of these leads to diseases

Fish and other species were perished for soiling and economy clearing in Tisza river at latest decades

Water pollution and remediation options for multi-source contaminations in freshwaters are happening sometimes, particularly in River Tisza after the cyanide and heavy metals spills of Romanian origin in 2000. The eco-toxicological effects, degradation and bioaccumulation rates of heavy metals have been followed in the next two years. Here the aim was to follow the bioremediation program and present the renewed economy of River Tisza. High concentration of arsenic, lead, mercury and cadmium was measured in the periphython and sediments samples both of the Rivers Tisza and Szamos. International rehabilitation program was planned and accomplished. Here we demonstrate the renewed economy of Tisza with living flora and fauna after 20 years, and the birth of day of May-fly again

Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems—the only DNA transposons currently employed in clinical trials—during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window

Cross-species interaction between rapidly evolving telomere-specific Drosophila proteins

Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction

Reversible binding of divalent cations to Ductin protein assemblies—A putative new regulatory mechanism of membrane traffic processes

Ductins are a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices. The active forms of the Ductins are membranous ring- or star-shaped oligomeric assemblies and they provide various pore, channel, gap-junction functions, assist in membrane fusion processes and also serve as the rotor c-ring domain of V-and F-ATPases. All functions of the Ductins have been reported to be sensitive to the presence of certain divalent metal cations (Me2+), most frequently Cu2+ or Ca2+ ions, for most of the better known members of the family, and the mechanism of this effect is not yet known. Given that we have earlier found a prominent Me2+ binding site in a well-characterised Ductin protein, we hypothesise that certain divalent cations can structurally modulate the various functions of Ductin assemblies via affecting their stability by reversible non-covalent binding to them. A fine control of the stability of the assembly ranging from separated monomers through a loosely/weakly to tightly/strongly assembled ring might render precise regulation of Ductin functions possible. The putative role of direct binding of Me2+ to the c-ring subunit of active ATP hydrolase in autophagy and the mechanism of Ca2+-dependent formation of the mitochondrial permeability transition pore are also discussed

Efficient and Specific Targeting of Polycomb Group Proteins Requires Cooperative Interaction between Grainyhead and Pleiohomeotic

Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. Here we show that the grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, we propose that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity