Molecular Analysis of N6-Methyladenine Patterns in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e Nuclear DNA

We have cloned two DNA fragments containing 5\u27-GATC-3\u27 sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns

Transformation of \u3cem\u3eTetrahymena thermophila\u3c/em\u3e with Hypermethylated rRNA Genes

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5\u27-NAT-3\u27, the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations

DNA methylation in ciliates: implications in differentiation processes

Much experimental evidence on the role of DNA methylation in gene expression has been reported. Here we review reports on DNA methylation in ciliated protozoa, emphasizing its implications in cell differentiation processes. Both types of methylated bases (adenine and cytosine) can be found in macronuclear DNA. The division cycle and conjugation have been studied with regard to adenine methylation, and several different functions have been assigned to the methylation changes detected in these processes. Cytosine methylation changes were analyzed during stomatogenesis of Paramecium and encystment of Colpoda inflata. A comparative analysis with other similar microbial eukaryotic differentiation processes is carried out

A lexicon of DNA modifications: their roles in embryo development and the germline

5-methylcytosine (5mC) on CpG dinucleotides has been viewed as the major epigenetic modification in eukaryotes for a long time. Apart from 5mC, additional DNA modifications have been discovered in eukaryotic genomes. Many of these modifications are thought to be solely associated with DNA damage. However, growing evidence indicates that some base modifications, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and N6-methadenine (6mA), may be of biological relevance, particularly during early stages of embryo development. Although abundance of these DNA modifications in eukaryotic genomes can be low, there are suggestions that they cooperate with other epigenetic markers to affect DNA-protein interactions, gene expression, defense of genome stability and epigenetic inheritance. Little is still known about their distribution in different tissues and their functions during key stages of the animal lifecycle. This review discusses current knowledge and future perspectives of these novel DNA modifications in the mammalian genome with a focus on their dynamic distribution during early embryonic development and their potential function in epigenetic inheritance through the germ line

Nuclear events during conjugation in the poorly studied model ciliate Paramecium jenningsi

Ciliated protists are highly differentiated unicellular eukaryotes that possess special sexual processes (conjugation and autogamy) that rely on their unique nuclear dimorphism, i.e., the presence of both a germline micronucleus (MIC) and a somatic macronucleus (MAC) in a single cell. The sexual processes show a high diversity in different ciliates. To better understand the differentiation and evolution of sexual processes in closely related species, we investigated the nuclear events during conjugation in Paramecium jenningsi strain GZ, a poorly studied close relative of the well-known P. aurelia complex. The main results include: (1) the conjugation process takes about 48 ​h, including three prezygotic divisions (meiosis I, II, and mitosis) and three postzygotic divisions; (2) the MICs are dominated by the “parachute” phase at the prophase of meiosis I; (3) after meiosis II, a variable number of nuclei undergo the third prezygotic division, and the two products near the paroral cone become the genetically identical migratory and stationary pronuclei; (4) the synkaryon divides three times to form the MIC and the MAC anlagen, but only one nuclear product of the first postzygotic division completes the next two divisions; and (5) an extra cell division is required to complete the last step of conjugation, during which two MIC anlagen undergo mitotic division while two MAC anlagen are distributed between the daughter cells without division. The comparison of the nuclear events during conjugation in representatives of the class Oligohymenophorea reveals that usually there are three prezygotic divisions and a variable number of postzygotic divisions. However, the number of selected and differentiated nuclei after each division differs among species. This study provided a new model ciliate for further investigations of nuclear selection and differentiation as well as nuclear morphology during meiosis

Regulation of transposable elements by DNA modifications

Wellcome Trust and the Royal Society (101225/Z/13/Z)People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement number 608765

A highly condensed genome without heterochromatin : orchestration of gene expression and epigenomics in Paramecium tetraurelia

Epigenetic regulation in unicellular ciliates can be as complex as in metazoans and is well described regarding small RNA (sRNA) mediated effects. The ciliate Paramecium harbors several copies of sRNA-biogenesis related proteins involved in genome rearrangements resulting in chromatin alterations. The global chromatin organization thereby is poorly understood, and unusual characteristics of the somatic nucleus, like high polyploidy, high genome coding density, and absence of heterochromatin, ought to call for complex regulation to orchestrate gene expression. The present study characterized the nucleosomal organization required for gene regulation and proper Polymerase II activity. Histone marks reveal broad domains in gene bodies, whereas intergenic regions are nucleosome free. Low occupancy in silent genes suggests that gene inactivation does not involve nucleosome recruitment. Thus, Paramecium gene regulation counteracts the current understanding of chromatin biology. Apart from global nucleosome studies, two sRNA binding proteins (Ptiwis) classically associated with transposon silencing were investigated in the background of transgene-induced silencing. Surprisingly, both Ptiwis also load sRNAs from endogenous loci in vegetative growth, revealing a broad diversity of Ptiwi functions. Together, the studies enlighten epigenetic mechanisms that regulate gene expression in a condensed genome, with Ptiwis contributing to transcriptome and chromatin dynamics.Epigenetische Regulation kann in einzelligen Ciliaten so komplex sein wie in Vielzellern und wurde umfassend angesichts kleiner RNA (sRNA)-vermittelter Effekte untersucht. Der Ciliat Paramecium besitzt mehrere Kopien sRNA-Biogenese assoziierter Proteine, die an Genomprozessierungen und resultierenden Chromatinänderungen beteiligt sind. Die globale Organisation des Chromatins ist dabei kaum verstanden und obskure Eigenschaften des somatischen Kerns, wie hohe Polyploidie, Kodierungsdichte und Fehlen von Heterochromatin, sollten eine komplexe Regulation zur Steuerung der Genexpression erfordern. Die vorliegende Studie charakterisiert die Chromatinorganisation, die für die Genregulation und Polymerase II Aktivität notwendig ist. Histonmodifikationen zeigen breite Verteilungen in Genen, während intergenische Regionen Nukleosomen-frei sind. Ein Stilllegen von Genen scheint ohne die Rekrutierung von Nukleosomen zu erfolgen, womit die Genregulation in Paramecium dem aktuellen Verständnis der Chromatinbiologie widerspricht. Neben Nukleosomenstudien wurden zwei sRNA-bindende Proteine (Ptiwis), die klassisch mit Transposon-Silencing assoziiert sind, im Hintergrund des Transgeninduzierten Silencings untersucht. Überraschenderweise laden Ptiwis sRNAs von endogenen Loci im vegetativen Wachstum, was vielfältige Ptiwi-Funktionen offenbart. Die Studien zeigen epigenetische Mechanismen zur Genregulation in einem kompakten Genom, wobei Ptiwis zur Transkriptom- und Chromatindynamik beitragen

Evolutionary origin and hidden genetic diversity of paramecium (ciliophora: oligohymenophorea)

Members of the genus Paramecium (Ciliophora, Oligohymenophorea) are widespread ciliates able to colonize many aquatic brackish and freshwater environments. They are currently divided in 5 subgenera encompassing 19 valid species, including Paramecium multimicronucleatum, Paramecium caudatum, Paramecium aurelia (complex) and Paramecium bursaria, which are considered cosmopolitan organisms. Despite that many Paramecium species have been historically and extensively used as model organisms in research fields ranging from genomics to conservation biology, their origin and populational structure remains elusive. Here, two datasets (18S rDNA and cytochrome oxidase subunit I [COI]) including sequences from all 5 subgenera and from most of the accepted species, we used the 18S rDNA to reconstruct a time-calibrated tree of Paramecium to elucidate the evolutionary origin of extant accepted Paramecium species and to evaluate intra-genus phylogenetic relationships. The COI dataset was used to evaluate, through species delimitation algorithms (PTP and mPTP) the existence of independent evolutionary unities (IEUs) within accepted Paramecium species. Our data rises more evidences supporting the idea that most accepted Paramecium species may be, in fact complexes of species, and that the diversity of cryptic species may be higher than previously though; the identified IEUs are not spatially structured, actually, many of than co-occurs over vast geographical areas, indicating that intercontinental distances are not barriers for IEUs` dispersion and gene flow, and other eco-physiological features may be responsible to their distribution.Paramecium são seres vivos, microscópicos, que podem ser encontrados em vários ambientes aquáticos, tanto de água doce quanto salgada. Eles são seres unicelulares, ou seja, o seu corpo é formado apenas de uma célula, e é todo recoberto de cílios, alimentam-se de bactérias, algas, matéria em decomposição, pois eles não conseguem produzir seu próprio alimento. Estes seres vivos são muito abundantes, distribuídos entre 19 espécies válidas, e bastante utilizados como modelo de estudo em vários campos de pesquisa. Porém, questões sobre quando esses organismos surgiram, e como sua estrutura populacional se encontra ainda não estão muito claras para os pesquisadores. Então, os objetivos deste trabalho foram identificar quando estes seres vivos surgiram, e como essa estrutura populacional encontra-se organizada. Para isso, utilizamos sequencias de DNA, depositadas em bancos de dados próprios para esse fim, que juntamente com programas computacionais e fósseis datados previamente conhecidos, permitem estabelecer este surgimento. Encontramos que estes seres vivos surgiram há cerca de 300 milhões de anos atrás, e que cada uma de suas espécies, tiveram uma data distinta de surgimento. Além disso, certamente existam bem mais espécies do que é conhecido atualmente, e questões ambientais, bem como questões fisiológicas destes seres vivos, é o que provavelmente influencia na sua distribuição.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

HIF-1α Metabolic Pathways in Human Cancer

Oxygen is directly involved in many key pathophysiological processes. Oxygen deficiency, also known as hypoxia, could have adverse effects on mammalian cells, with ischemia in vital tissues being the most significant (Michiels C. Physiological and pathological responses to hypoxia. Am J Pathol 164(6): 1875-1882, 2004); therefore, timely adaptive responses to variations in oxygen availability are essential for cellular homeostasis and survival. The most critical molecular event in hypoxic response is the activation and stabilization of a transcriptional factor termed hypoxia-induced factor-1 (HIF-1) that is responsible for the upregulation of many downstream effector genes, collectively known as hypoxia-responsive genes. Multiple key biological pathways such as proliferation, energy metabolism, invasion, and metastasis are governed by these genes; thus, HIF-1-mediated pathways are equally pivotal in both physiology and pathology.As we gain knowledge on the molecular mechanisms underlying the regulation of HIF-1, a great focus has been placed on elucidating the cellular function of HIF-1, particularly the role of HIF-1 in cancer pathogenesis pathways such as proliferation, invasion, angiogenesis, and metastasis. In cancer, HIF-1 is directly involved in the shift of cancer tissues from oxidative phosphorylation to aerobic glycolysis, a phenomenon known as the Warburg effect. Although targeting HIF-1 as a cancer therapy seems like an extremely rational approach, owing to the complex network of its downstream effector genes, the development of specific HIF-1 inhibitors with fewer side effects and more specificity has not been achieved. Therefore, in this review, we provide a brief background about the function of HIF proteins in hypoxia response with a special emphasis on the unique role played by HIF-1α in cancer growth and invasiveness, in the hypoxia response context