Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

Eukaryotes can have thousands of 45S ribosomal RNA (rRNA) genes, many of which are silenced during development. Using fluorescence-activated sorting techniques, we show that active rRNA genes in Arabidopsis thaliana are present within sorted nucleoli, whereas silenced rRNA genes are excluded. DNA methyltransferase (met1), histone deacetylase (hda6), or chromatin assembly (caf1) mutants that disrupt silencing abrogate this nucleoplasmic–nucleolar partitioning. Bisulfite sequencing data indicate that active nucleolar rRNA genes are nearly completely demethylated at promoter CGs, whereas silenced genes are nearly fully methylated. Collectively, the data reveal that rRNA genes occupy distinct but changeable nuclear territories according to their epigenetic state.     

Human active X-specific DNA methylation events showing stability across time and tissues

The phenomenon of X chromosome inactivation in female mammals is well characterised and remains the archetypal example of dosage compensation via monoallelic expression. The temporal series of events that culminates in inactive X-specific gene silencing by DNA methylation has revealed a ‘patchwork'' of gene inactivation along the chromosome, with approximately 15% of genes escaping. Such genes are therefore potentially subject to sex-specific imbalance between males and females. Aside from XIST, the non-coding RNA on the X chromosome destined to be inactivated, very little is known about the extent of loci that may be selectively silenced on the active X chromosome (Xa). Using longitudinal array-based DNA methylation profiling of two human tissues, we have identified specific and widespread active X-specific DNA methylation showing stability over time and across tissues of disparate origin. Our panel of X-chromosome loci subject to methylation on Xa reflects a potentially novel mechanism for controlling female-specific X inactivation and sex-specific dimorphisms in humans. Further work is needed to investigate these phenomena.     

Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.     

Histone methyltransferases regulating rRNA gene dose and dosage control in Arabidopsis

Eukaryotes have hundreds of nearly identical 45S ribosomal RNA (rRNA) genes, each encoding the 18S, 5.8S, and 25S catalytic rRNAs. Because cellular demands for ribosomes and protein synthesis vary during development, the number of active rRNA genes is subject to dosage control. In genetic hybrids, one manifestation of dosage control is nucleolar dominance, an epigenetic phenomenon in which the rRNA genes of one progenitor are repressed. For instance, in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, the A. thaliana-derived rRNA genes are selectively silenced. An analogous phenomenon occurs in nonhybrid A. thaliana, in which specific classes of rRNA gene variants are inactivated. An RNA-mediated knockdown screen identified SUVR4 {SUPPRESSOR OF VARIEGATION 3-9 [SU(VAR)3-9]-RELATED 4} as a histone H3 Lys 9 (H3K9) methyltransferase required for nucleolar dominance in A. suecica. H3K9 methyltransferases are also required for variant-specific silencing in A. thaliana, but SUVH5 [SU(VAR)3-9 HOMOLOG 5] and SUVH6, rather than SUVR4, are the key activities in this genomic context. Mutations disrupting the H3K27 methyltransferases ATXR5 or ATXR6 affect which rRNA gene variants are expressed or silenced, and in atxr5 atxr6 double mutants, dominance relationships among variants are reversed relative to wild type. Interestingly, these changes in gene expression are accompanied by changes in the relative abundance of the rRNA gene variants at the DNA level, including overreplication of the normally silenced class and decreased abundance of the normally dominant class. Collectively, our results indicate that histone methylation can affect both the doses of different variants and their differential silencing through the choice mechanisms that achieve dosage control.     

Mapping of rRNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe

Summary The major rRNA genes of the fission yeast Schizosaccharomyces pombe were mapped on chromosome III by plasmid integration. The integration vector YIp33 containing S. cerevisiae LEU2 gene was combined with the S. pombe rDNA. Since LEU2 complements S. pombe leu1 deficiency, it could be used as the genetic marker for integration. The 10.4 kb rDNA repeat contained ARS sequence, and therefore 2.4 kb and 0.7 kb subfragments not containing ARS were subcloned into YIp33 and transformed leu1 S. pombe cells to Leu⁺. Genetic analyses of the transformants indicated that the integrated rDNA resides in the long arm of the shortest chromosome III, tightly linked to ade5 (1.4 cM). This result is consistent with our previous finding that the DAPI-stained smallest chromosomes were associated with the nucleolus (Umesono et al. 1983).Abbreviations ARS autonomously replicating sequence - DAPI 4,6-diamidino-2-phenylindole - kb kilo base pairs - rDNA DNA segment containing ribosomal RNA genes - rRNA ribosomal RNA     

Isolation,X location and activity of the marsupial homologue of <Emphasis Type="Italic">SLC16A2</Emphasis>, an <Emphasis Type="Italic">XIST</Emphasis>-flanking gene in eutherian mammals

X chromosome inactivation (XCI) achieves dosage compensation between males and females for most X-linked genes in eutherian mammals. It is a whole-chromosome effect under the control of the XIST locus, although some genes escape inactivation. Marsupial XCI differs from the eutherian process, implying fundamental changes in the XCI mechanism during the evolution of the two lineages. There is no direct evidence for the existence of a marsupial XIST homologue. XCI has been studied for only a handful of genes in any marsupial, and none in the model kangaroo Macropus eugenii (the tammar wallaby). We have therefore studied the sequence, location and activity of a gene SLC16A2 (solute carrier, family 16, class A, member 2) that flanks XIST on the human and mouse X chromosomes. A BAC clone containing the marsupial SLC16A2 was mapped to the end of the long arm of the tammar X chromosome and used in RNA FISH experiments to determine whether one or both loci are transcribed in female cells. In male and female cells, only a single signal was found, indicating that the marsupial SLC16A2 gene is silenced on the inactivated X.     

Multi-chromosomal location of ribosomal RNA genes and heterochromatin association in brown trout

The ribosomal rRNA genes have been mapped by fluorescentin situ hybridization (FISH) to brown trout chromosomes. One major NOR chromosome pair and 8 novel minor NOR chromosome pairs have been found. Both major and minor NORs were closely related to polymorphic heterochromatin, as revealed by FISH and C-banding. These results are discussed with respect to NOR expression, the relationship between rDNA and heterochromatin, and evolutionary aspects.     

Detection of variation in the ribosomal RNA gene clusters by a modified fluorescencein situ hybridization method

Summary Physical mapping of genes by fluorescencein situ hybridization (FISH) has become routine using fluorescein isothiocyanate (FITC) for probe detection and propidium iodide (PI) for chromosome staining. We have modified this conventional FISH method in a way that utilizes Texas red (TR) for signal detection and quinacrine mustard (QM) for chromosome banding. Using this Texas red and quinacrine (TRQ) method, we were able to identify individual acrocentric chromosomes with varying degrees of ribosomal RNA gene clusters. Two acrocentric chromosomes were found to carry extremely small number of rRNA gene copies as compared to the other eight counterparts in human diploid lymphoblastoid cell line GM00130B. Thus, the TRQ method allows one to probe for a specific sequence while identifying individual chromosomes and will be powerful for the chromosomal localization of various genes.     

Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.     

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.     

Chromosomal mapping of the uracil permease gene of Saccharomyces cerevisiae

Summary The gene FUR4, coding for the uracil permease in Saccharomyces cerevisiae, was mapped on chromosome II, at a distance of 7.8 cM from the centromere on the right arm of the chromosome. In a first step, we used the chromosome loss mapping method developed by Falco and Botstein (1983) to determine on which chromosome the gene mapped. After the observation that FUR4 was closely linked to GAL10, one of the three genes forming the gal cluster (Bassel and Mortimer 1971), we could determine precisely the position of the gene on chromosome II.     

Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication

A functional screen of an adenovirus-delivered shRNA library that targets ∼ 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.     

Silver bands in chronic granulocytic leukemia. II. The Philadelphia chromosome

To test if rRNA gene activity in the Philadelphia (Ph) chromosome nucleolar organizing regions (NOR) differs from other NOR we scored silver banding patterns in leukemic cells of 32 patients (one patient was sampled twice) in the chronic phase of chronic granulocytic leukemia. The average number of bands per NOR in banded cells of these patients was 0.40 +/- 0.06 for the Ph NOR, not significantly different from the 0.41 +/- 0.02 per D chromosome NOR or the 0.41 +/- 0.04 per G. Across the 33 cases, the number of different Ph NOR banded in at least one cell (23 of 33, 70%) was also closely similar to the cumulative maximum number of chromosome D NOR banded per cell (137 of 198 banded, 69%) and G NOR banded (67 of 99, 67%). We conclude that the activity of rRNA genes in Ph NOR is closely similar to that of the other nine NOR in each CGL cell. In situ hybridization of 3H-labeled complementary rRNA to NOR of two patients revealed that the mean number of Ph NOR grains fell within the range of the other NOR. Also, grain distributions across the NOR of leukemic cells closely matched those of stimulated lymphocytes. Thus, a limited sampling has shown no evidence for consistent differences or changes in the rRNA gene number of Ph NOR.