RESUMEN
Histone H3K27me3 modification is an important regulator for development and gene expression. In Tetrahymena thermophila, the complex chromatin dynamics of H3K27me3 marks during nuclear development suggested that an H3K27me3 demethylase might exist. Here, we report an H3K27me3 demethylase homolog, JMJ1, in Tetrahymena. During conjugation, JMJ1 expression is upregulated and the protein is localized first in the parental macronucleus and then in the new macronucleus. In conjugating cells, knockdown of JMJ1 expression resulted in a severe reduction in the production of progeny, suggesting that JMJ1 is essential for Tetrahymena conjugation. Furthermore, knockdown of JMJ1 resulted in increased H3K27 trimethylation in the new macronucleus and reduced transcription of genes related to DNA elimination, while the DNA elimination process was also partially blocked. Knockdown of the H3K27 methyltransferase EZL2 but not that of EZL1 partially restored progeny production in JMJ1-knockdown cells and reduced abnormal H3K27me3 accumulation in the new macronucleus. Taken together, these results demonstrate a critical role for JMJ1 in regulating H3K27me3 during conjugation and the importance of JMJ1 in regulating gene expression in the new macronucleus but not in regulating the formation of heterochromatin associated with programmed DNA deletion.
Asunto(s)
Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/metabolismo , Secuencia de Aminoácidos , Western Blotting , Cloruro de Cadmio/farmacología , Inmunoprecipitación de Cromatina , Rotura Cromosómica , Biología Computacional , Conjugación Genética , ADN Protozoario/genética , ADN Protozoario/metabolismo , Técnicas de Silenciamiento del Gen , Heterocromatina/genética , Heterocromatina/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Macronúcleo/enzimología , Macronúcleo/genética , Macronúcleo/metabolismo , Metilación , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Filogenia , Proteínas Protozoarias/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tetrahymena thermophila/enzimología , Tetrahymena thermophila/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (~70 kb) plus a ~5-kb linear plasmid bearing mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps- 2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence. The increased size of the Oxytricha mitochondrial genome relative to other ciliates is primarily a consequence of terminal expansions, rather than the retention of ancestral mitochondrial genes. Successive segmental duplications, visible in one of the two Oxytricha mitochondrial subterminal regions, appear to have contributed to the genome expansion. Consistent with pseudogene formation and decay, the subtermini possess shorter, more loosely packed open reading frames than the remainder of the genome. The mitochondrial plasmid shares a 251-bp region with 82% identity to the mitochondrial chromosome, suggesting that it most likely integrated into the chromosome at least once. This region on the chromosome is also close to the end of the most terminal member of a series of duplications, hinting at a possible association between the plasmid and the duplications. The presence of mitochondrial telomeres on the mitochondrial plasmid suggests that such plasmids may be a vehicle for lateral transfer of telomeric sequences between mitochondrial genomes. We conjecture that the extreme divergence observed in ciliate mitochondrial genomes may be due, in part, to repeated invasions by relatively error-prone DNA polymerase-bearing mobile elements.
Asunto(s)
Genoma Mitocondrial/genética , Genoma de Protozoos/genética , Oxytricha/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas/genética , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/genética , Genes Protozoarios/genética , Código Genético/genética , Macronúcleo/enzimología , Macronúcleo/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Plásmidos/genética , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN Ribosómico/genética , ARN de Transferencia/genética , Duplicaciones Segmentarias en el Genoma/genética , Sintenía/genéticaRESUMEN
The ciliate Paramecium tetraurelia must eliminate approximately 60,000 short sequences from its genome to generate uninterrupted coding sequences in its somatic macronucleus. In this issue of Genes & Development, Baudry and colleagues (pp. 2478-2483) identify the protein that excises these noncoding sequences: a domesticated piggyBac transposase that has been adapted to remove what are likely the remnants of transposon insertions. This new study reveals how addition of a transposase to small RNA-directed silencing machinery can guide major genome reorganization.
Asunto(s)
ADN Protozoario/metabolismo , Genoma/genética , Macronúcleo/enzimología , Paramecium tetraurelia/enzimología , Paramecium tetraurelia/genética , Transposasas/metabolismo , Animales , Interferencia de ARN/fisiologíaRESUMEN
Class II polypeptide release factor (eRF3), a ribosome and eRF1-dependent GTPase, is an important factor, which acts cooperatively with eRF1 to promote hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA in process of termination of protein synthesis. We prepared antibodies against eRF3 of Euplotes octocarinatus, and performed localization studies by immunoelectron microscopy in the ciliate. Our results indicate that eRF3 is present both in the cytoplasm and the two types of nuclei of this organism. The functions of eRF3 in these nuclei were analyzed by RNA interference methods. The nuclei loose their shape in eRF3 gene-interfered Euplotes cells, suggesting that eRF3 is probably involved in the morphological organization of nuclei. This suggests that eRF3 is a multifunctional protein with roles additionals to its function in the process of termination of protein synthesis.
Asunto(s)
Núcleo Celular/enzimología , Euplotes/enzimología , Factores de Terminación de Péptidos/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Línea Celular , Núcleo Celular/ultraestructura , Forma del Núcleo Celular , Citoplasma/enzimología , Euplotes/ultraestructura , Macronúcleo/enzimología , Micronúcleo Germinal/enzimología , Microscopía Inmunoelectrónica , Biosíntesis de Proteínas , Proteínas Protozoarias/biosíntesis , Interferencia de ARNRESUMEN
Newly synthesized histones are acetylated prior to their deposition into nucleosomes. Following nucleosome formation and positioning, they are rapidly deacetylated, an event that coincides with further maturation of the chromatin fiber. The histone deacetylases (HDACs) used for histone deposition and de novo chromatin formation are poorly understood. In the ciliate Tetrahymena thermophila, transcription-related deacetylation in the macronucleus is physically separated from deposition-related deacetylation in the micronucleus. This feature was utilized to identify an HDAC named Thd2, a class II HDAC that acts on newly synthesized histones to remove deposition-related acetyl moieties. The THD2 transcript is alternatively spliced, and the major form contains a putative inositol polyphosphate kinase (IPK) domain similar to Ipk2, an enzyme that promotes chromatin remodeling by SWI/SNF remodeling complexes. Cells lacking Thd2, which retain deposition-related acetyl moieties on new histones, exhibit chromatin and cytological phenotypes indicative of a role for Thd2 in chromatin maturation, including the proteolytic processing of histone H3.
Asunto(s)
Histona Desacetilasas/metabolismo , Histonas/metabolismo , Tetrahymena thermophila/enzimología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Replicación del ADN , Macronúcleo/enzimología , Micronúcleo Germinal/enzimología , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Tetrahymena thermophila/metabolismoRESUMEN
Proteins containing a Tudor domain and domains homologous to staphylococcal nucleases are found in a number of eukaryotes. These "Tudor nucleases" have been found to be associated with the RNA-induced silencing complex (A. A. Caudy, R. F. Ketting, S. M. Hammond, A. M. Denli, A. M. Bathoorn, B. B. Tops, J. M. Silva, M. M. Myers, G. J. Hannon, and R. H. Plasterk, Nature 425:411-414, 2003). We have identified two Tudor nuclease gene homologs, TTN1 and TTN2, in the ciliate Tetrahymena thermophila, which has two distinct small-RNA pathways. Characterization of single and double KOs of TTN1 and TTN2 shows that neither of these genes is essential for growth or sexual reproduction. Progeny of TTN2 KOs and double knockouts occasionally show minor defects in the small-RNA-guided process of DNA deletion but appear to be normal in hairpin RNA-induced gene silencing, suggesting that Tudor nucleases play only a minor role in RNA interference in Tetrahymena. Previous studies of Tetrahymena have shown that inserted copies of the neo gene from Escherichia coli are often deleted from the developing macronucleus during sexual reproduction (Y. Liu, X. Song, M. A. Gorovsky, and K. M. Karrer, Eukaryot. Cell 4:421-431, 2005; M. C. Yao, P. Fuller, and X. Xi, Science 300:1581-1584, 2003). This transgene deletion phenomenon is hypothesized to be a form of genome defense. Analysis of the Tudor nuclease mutants revealed exceptionally high rates of deletion of the neo transgene at the TTN2 locus but no deletion at the TTN1 locus. When present in the same genome, however, the neo gene is deleted at high rates even at the TTN1 locus, further supporting a role for trans-acting RNA in this process. This deletion is not affected by the presence of the same sequence in the macronucleus, thus providing a counterargument for the role of the macronuclear genome in specifying all sequences for deletion.
Asunto(s)
Desoxirribonucleasas/genética , Reordenamiento Génico/genética , Genes Protozoarios , Tetrahymena thermophila/enzimología , Tetrahymena thermophila/genética , Animales , Secuencia de Bases , Desoxirribonucleasas/química , Farmacorresistencia Microbiana , Eliminación de Gen , Macronúcleo/efectos de los fármacos , Macronúcleo/enzimología , Datos de Secuencia Molecular , Neomicina/farmacología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Interferencia de ARN , Reproducción/efectos de los fármacos , Eliminación de Secuencia , Tetrahymena thermophila/efectos de los fármacos , Tetrahymena thermophila/crecimiento & desarrollo , TransgenesRESUMEN
Class I histone deacetylases (HDACs) participate in the regulation of DNA-templated processes such as transcription and replication. Members of this class can act locally at specific sites, or they can act more globally, contributing to a baseline acetylation state, both of which actions may be important for genome maintenance and organization. We previously identified a macronuclear-specific class I HDAC in Tetrahymena thermophila called Thd1p, which is expressed early in the development of the macronucleus when it initially becomes transcriptionally active. To test the idea that Thd1p is important for global chromatin integrity in an active macronucleus, Tetrahymena cells reduced in expression of Thd1p were generated. We observed phenotypes that indicated loss of chromatin integrity in the mutant cells, including DNA fragmentation and extrusion of chromatin from the macronucleus, variable macronuclear size and shape, enlarged nucleoli, and reduced phosphorylation of histone H1 from bulk chromatin. Macronuclei in mutant cells also contained more DNA. This observation suggests a role for Thd1p in the control of nuclear DNA content, a previously undescribed role for class I HDACs. Together, these phenotypes implicate Thd1p in the maintenance of macronuclear integrity in multiple ways, probably through site-specific changes in histone acetylation since no change in the acetylation levels of bulk histones was detected in mutant cells.
Asunto(s)
Histona Desacetilasas/fisiología , Macronúcleo/enzimología , Proteínas Protozoarias/fisiología , Tetrahymena thermophila/enzimología , Acetilación , Animales , Cromatina/metabolismo , Replicación del ADN , ADN Protozoario/metabolismo , Eliminación de Gen , Histona Desacetilasas/genética , Histonas/metabolismo , Macronúcleo/genética , Macronúcleo/ultraestructura , Fosforilación , Proteínas Protozoarias/genética , Tetrahymena thermophila/genéticaRESUMEN
A key characteristic of apoptosis is its regulated nuclear degradation. Apoptosis-like nuclear degradation also occurs in the ciliated unicellular organism, Tetrahymena thermophila. Chromatin of the macronucleus undergoes massive condensation, a process that can be blocked by caspase inhibitors. The nucleus becomes TUNEL-positive, and its DNA is cleaved into nucleosome-sized fragments. In a matter of hours the macronucleus is completely degraded, and disappears. The condensed nucleus sequesters acridine orange, which means that it might become an acidic compartment. We therefore asked whether lysosomal bodies fuse with the condensed macronucleus to form an autophagosome. We monitored acid phosphatase (AP) activity, which is associated with lysosomal bodies but is not found in normal nuclei. We find that after the macronucleus condenses AP activity is localized in cap-like structures at its cortex. Later, after the degrading macronucleus loses much of its DNA, acid phosphatase deposits appear deeper within the nucleus. We conclude that although macronuclear elimination is initiated by an apoptosis-like mechanism, its final degradation may be achieved through autophagosomy.