Linker Histone H1 Regulates Specific Gene Expression but Not Global Transcription In Vivo

AbstractIn a linker histone H1 knockout strain (ΔH1) of Tetrahymena thermophila, the number of mature RNAs produced by genes transcribed by pol I and pol III and of most genes transcribed by pol II remains unchanged. However, H1 is required for the normal basal repression of a gene (ngoA) in growing cells but is not required for its activated expression in starved cells. Surprisingly, H1 is required for the activated expression of another gene (CyP) in starved cells but not for its repression in growing cells. Thus, H1 does not have a major effect on global transcription but can act as either a positive or negative gene-specific regulator of transcription in vivo

Proteomic Analysis of Core Histones and Their Variants in Tetrahymena Thermophila

Several histone chaperones including Nasp, Asf1, Caf1 and Hira have been identified to function in the transport and assembly of newly synthesized histones. To characterize histone transport machinery and chromatin assembly proteins in ciliate protozoan Tetrahymena thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of core histone H2A, variants Hv1 and H3.3 as well as linker histone MLH1. I found that H2A co-purifies with putative Spt16Tt and Pob3Tt subunits of the T. thermophila FACT complex. Proteomic analysis of Hv1 indicated that it co-purifies with an Importinβ3, suggesting a possible mechanism of targeting Hv1 specifically to transcriptionally active macronucleus. My data also indicated that H2A, Hv1 and H3.3 co-purify with putative PARP1 and PARP2 proteins suggesting that ribosylation of histones might have a critical role in the chromatin maintenance

Microarray Analyses of Gene Expression during the Tetrahymena thermophila Life Cycle

The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.Of the approximately 27,000 predicted open reading frames, transcripts homologous to only approximately 5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions

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

Analysis of the somatic and germline genomes of the ciliate Blepharisma stoltei

Ciliaten sind prototypische, üblicherweise einzellige Eukaryoten mit getrennten Keimbahn- und somatischen Zellkernen. Das somatische Genom entsteht aus dem Keimbahngenom durch einen Prozess der Transposase-vermittelten DNA-Eliminierung und Genom-Neuordnung während der sexuellen Fortpflanzung. Aktuelle Modelle für die Reorganisation des Genoms bei Wimpertierchen gehen davon aus, dass kleine RNAs während der sexuellen Fortpflanzung in den sich entwickelnden somatischen Kern transportiert werden und Transposasen dabei helfen, keimlinienspezifische Sequenzen zu identifizieren und auszuschneiden. Diese Sequenzen, die so genannten intern eliminierten Sequenzen (IES), und ihre Exzisasen werden von einer Maschinerie begleitet, die ihre Entfernung durchführt. Dazu gehören Dicer-ähnliche und Piwi/Argonaute-Proteine, die kleine RNAs erzeugen und transportieren, sowie Proteine, die das Chromatin verändern und die DNA für die Exzision zugänglich machen. Blepharisma gehört zu einer früh divergiereden Klasse von Ciliaten, die als Heterotrichea bekannt sind. Obwohl die Reorganisation des Genoms bei später divergierenden Ciliaten wie den oligohymenophoren Ciliaten Tetrahymena und Paramecium und den spirotrichen Oxytricha untersucht wurde, gibt es deutliche Unterschiede in der Art und Weise, wie sie dies tun. Die Untersuchung dieses Prozesses in einem früh divergiereden Ciliaten wie Blepharisma ist ein wichtiger Beitrag zum Verständnis, wie konserviert die verschiedenen Elemente der Genom- Reorganisationsmaschinerie unter Ciliaten sind. Diese Arbeit bietet den ersten Blick aus genomischer Sicht auf die verschiedenen Teilnehmer und mutmaßlichen Mechanismen der Genomreorganisation in Blepharisma. Mittels Long-Read-Sequenzierung und Annotationsmethoden, die auf die atypischen Genomeigenschaften von Blepharisma zugeschnitten sind, wurden annotierte Referenzgenome für die somatischen und Keimbahnkerne von Blepharisma stoltei (Stamm ATCC 30299) erstellt. Das somatische Genom von B. stoltei ist kompakt (41 Mb), gen-dicht (25710 Gene) und enthält kurze, 15-16 Nukleotide umfassende spliceosomale Introns. Wir haben Schlüsselkomponenten identifiziert, die an der Reorganisation des Genoms im somatischen Genom von Blepharisma beteiligt sind, und sie mit denen der Modell-Ciliaten Paramecium, Tetrahymena und Oxytricha verglichen. Es wurden vier Transposase-Familien gefunden, die in den somatischen und Keimbahn-Genomen kodiert sind, nämlich die PiggyBac-,Tc1/Mariner-, Mutator- und Merlin-Familien. Es ist bekannt, dass PiggyBac-Transposasen die wichtigsten Transposasen sind, die in den Modell-Ciliaten Paramecium und Tetrahymena an der Reorganisation des Genoms beteiligt sind, während sie in Oxytricha, wo vermutlich eine Transposase aus einer anderen Familie verwendet wird, gänzlich fehlen. In Paramecium koordinieren sechs somatisch kodierte PiggyBacs, die nicht zur Katalyse fähig sind, sowie ein katalytisch vollständiges Homolog, namens PiggyMac, die DNA-Exzision. Dies ähnelt der Situation in Blepharisma, wo es dreizehn Homologe der PiggyBac-Transposase gibt, von denen nur eine eine vollständige katalytische Triade besitzt und daher wahrscheinlich die primäre Exzisase ist. Die keimbahnbegrenzten genomischen Regionen von Blepharisma wurden ebenfalls charakterisiert. Die IES von Blepharisma haben zwei wesentliche Merkmale mit den IES von Paramecium gemeinsam, nämlich eine periodische Längenverteilung für kurze IES und überwiegend durch TA-Dinukleotide abgegrenzte IES-Grenzen. Wir haben auch eine Klasse von kleinen RNAs („small RNAs“ ) mit 24 Nukleotiden identifiziert, die mit fortschreitender Entwicklung in Blepharisma zunehmend den IESs zugeordnet werden. Diese Tendenzen ähneln denen, die in Paramecium und Tetrahymena beobachtet wurden, weshalb wir vorschlagen, dass es sich auch hier um so genannte "Scan"-RNAs (scnRNAs) handelt, die die IES-Exzision steuern. Die phylogenetische Analyse der PiggyBac-Homologe von Blepharisma hat gezeigt, dass sie einen gemeinsamen Ursprung mit den PiggyBac-Homologen von Paramecium und Tetrahymena haben, wobei letztere evolutionär stärker divergieren als Blepharisma und auf jüngeren Zweigen des phylogenetischen Stammbaums der Ciliaten zu finden sind. Mehrere Indizien aus diesen Studien deuten daher darauf hin, dass eine PiggyBac-Transposase höchstwahrscheinlich die wichtigste IES-Exzisase in Blepharisma ist und dass der letzte gemeinsame Vorfahre der Ciliaten ebenfalls diesen Transposasetyp besaß.Ciliates are prototypical, conventionally unicellular eukaryotes with separate germline and somatic nuclei. The somatic genome arises from the germline genome through a process of transposase-mediated DNA elimination and genome rearrangement during sexual reproduction. Current models for genome reorganization in ciliates posit that small RNAs are transported to the developing somatic nucleus during sexual reproduction, aiding transposases in identifying and excising germline-specific sequences. Accompanying these sequences, known as Internally Eliminated Sequences (IESs), and their excisases is the machinery to carry out their removal. This includes Dicer-like and Piwi/Argonaute proteins, which generate and transport small RNAs, as well as proteins that alter chromatin, and make DNA accessible for excision. The ciliate Blepharisma belongs to an early diverging class of ciliates known as the Heterotrichea. Though genome reorganization has been studied in later diverging ciliates such the oligohymenophorean ciliates Tetrahymena and Paramecium and the spirotrich Oxytricha there are pronounced differences in how they do so. Studying this process in an early diverging ciliate like Blepharisma is an important contribution to the understanding of how conserved the different elements of the genome reorganization machinery among ciliates are. This thesis provides the first look, from a genomic perspective, at the various participants and putative mechanisms of genome reorganization in Blepharisma. Annotated reference genomes for the somatic and germline nuclei of Blepharisma stoltei (strain ATCC 30299) were generated using long-read sequencing and annotation methods tailored to the atypical genome properties of Blepharisma. The B. stoltei somatic genome is compact (41 Mb), gene-dense (25710 genes) and contains short, 15-16 nucleotide spliceosomal introns. We identified key components involved in genome reorganization in the Blepharisma somatic genome and compared them with those of the model ciliates Paramecium, Tetrahymena and Oxytricha. Four transposase families were found encoded in the somatic and germline genomes, namely the PiggyBac, Tc1/Mariner, Mutator and Merlin families. PiggyBac transposases are known to be the main transposases involved in genome reorganization in the model ciliates Paramecium and Tetrahymena, but are entirely absent in Oxytricha, which is thought to use a transposase from another family. In Paramecium, six somatically encoded PiggyBacs incapable of catalysis, plus one catalytically complete homolog called the PiggyMac, coordinate DNA excision. This resembles the situation in Blepharisma, which has thirteen homologs of the PiggyBac transposase, only one of which has a complete catalytic triad and is hence likely to be the primary excisase. The germline-limited genomic regions of Blepharisma were also characterized. Blepharisma IESs share two key features with the IESs of Paramecium, namely a periodic length distribution for short IESs and predominantly TA-dinucleotide delineated IES boundaries. We also identified a class of 24-nucleotide small RNAs that increasingly map to IESs as development progresses in Blepharisma. These trends are similar to those observed in Paramecium and Tetrahymena, hence we propose that they are also so-called “scan” RNAs (scnRNAs) that guide IES excision. Phylogenetic analysis of the Blepharisma PiggyBac homologs showed that they share common ancestry with the PiggyBac homologs of Paramecium and Tetrahymena, where the latter are evolutionarily more divergent than Blepharisma and are located on more recently diverging branches of the ciliate phylogenetic tree. Several lines of evidence from these studies therefore indicate that a PiggyBac transposase is the most likely the main IES excisase in Blepharisma and that the last ciliate common ancestor also possessed this type of transposase

The Role of dsRNA in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena thermophila

The ciliate, Tetrahymena thermophila, like a handful of other eukaryotes, engages in massive genome reorganization known collectively as chromatin diminution. Part of this process involves large-scale DNA excision known as DNA elimination. Recent data has shown DNA elimination to be dependent on RNA interference: RNAi). Using T. thermophila, I have sought to determine the role of non-coding RNA: ncRNA) in RNAi-dependent DNA elimination through studies of DNA sequences that are to be eliminated called internal eliminated sequences: IESs) and through a conjugation-specific Dicer protein and its putative tandem dsRNA-binding motif: DSRM) protein partners. Studies of the R IES revealed the requirement of IES DNA for production of long, bidirectional ncRNA early in conjugation. This ncRNA is essential for IES excision in zygotic nuclei later in conjugation. The conjugation-specific Dicer homologue, DCL1, was shown to be required for production of a species of sRNA called scnRNAs from the long, bidirectional ncRNA from IESs. Knockouts of DCL1 displayed a loss of these scnRNAs as well as an increase in the long, bidirectional ncRNA precursors. A deficiency in these scnRNAs was sufficient to block modification of chromatin associated with IESs and prevent their rearrangement later in conjugation. Failure of DNA elimination caused DCL1 knockout cells to arrest before completion of conjugation. Further studies of the tandem DSRM-containing proteins, DRB2 and DRB1, revealed that neither are solely partners for DCL1 or any other Dicer protein but play other important roles during conjugation. Zygotic expression of DRB2 was shown to be essential for DNA elimination and completion of conjugation. Interaction with the chromo-domain containing protein, Pdd1p, by Drb2p implicates ncRNA or sRNA in later stages of conjugation after scnRNA production. Knockouts of the tandem DSRM-containing DRB1 caused higher numbers of cells to abort conjugation and therefore produce fewer progeny. Localization of this protein to the crescent micronucleus during prophase of meiosis I links DRB1 to a probable role in ensuring proper recombination during meiosis for haploid gamete production. All these studies suggest that ncRNA has many roles in conjugation-specific processes including RNAi-directed DNA elimination

Studies on the Histone Methyltransferase G9a

The size and complexity of eukaryotic genomes require that specific mechanisms exist for ensuring both the stability as well as the accessibility of DNA. One such mechanism is the association of DNA with histones to form chromatin, the physiological substrate of gene expression. An important means by which histones impact transcriptional activity is through site-specific enzymatic modification of the amino terminal histone “tailsâ€, which can alter the spectrum of chromatin-associated proteins and hence transcriptional states. Among the known modifications of histones, lysine methylation has been proposed to represent a relatively stable mark which might mediate stable activation or repression, depending upon the site modified. The immune system provides an ideal system in which to test the physiological functions of particular chromatin-modifying activities, since proper lymphocyte development and function requires integration of multiple signals, both cell autonomous and receptor-mediated, with complex DNA recombination reactions which are unique to lymphocytes. We have exploited these features to explore the possible functions of histone 3, lysine 3 (H3K9) methylation in the immune system through conditional inactivation of the H3K9-specific methyltransferases G9a and GLP. These studies demonstrate that G9a is essential for B cell development in the mouse, but is dispensable for T cell development. The defect in B lymphopoiesis in the absence of G9a is caused by a block in development at the pro-B cell stage, corresponding to the onset of immunoglobulin heavy chain recombination. The overall normal behavior of G9a-deficient peripheral B cells argues in favor of the specificity of this effect. Furthermore, through analysis of G9a and GLP protein sequences, we have identified a novel mechanism by which chromatin-modifying complexes can be regulated. We find that both G9a and GLP contain conserved H3K9-like motifs, on which the main biochemical features of H3K9 itself are recapitulated. Considering both the sequence and functional conservation between these sites and histones, we term these motifs in nonhistone proteins “histone mimicsâ€. Our initial analysis indicates that many chromatinassociated proteins potentially contain H3K9-type histone mimics, and that this phenomenon is therefore likely to be a general one

Genomic distribution of histone H1 in budding yeast (Saccharomyces cerevisiae) : yeast chromosome III

Includes bibliographical references.The linker histone HI binds to the nucleosome and is essential for the organization of nucleosomes into the 30-nm filament of the chromatin. This compaction of DNA has a well-characterized effect on DNA function. In Saccharomyces cerevisiae, HHO 1 encodes a putative linker histone with very significant homology to histone HI. In vitro chromatin assembly experiments with recombinant Hho 1 p have shown that it is able to complex with the dinucleosomes in a similar manner to histone HI. It has also been reported that disruption of HHOl has little affect on RNA levels. A longstanding issue concerns the location of Hho 1 p in the chromatin and studies have shown using immunoprecipitation technique with anti-HA antibody, that Hho 1 p shows a preferential binding to rDNA sequences. In this project we have tried to confirm the above results in wild type cells, using immunopurifi ed anti rHho 1 p antibody