RESUMO
The Arabidopsis chromosomes contain conspicuous heterochromatin domains comprising the repetitive 45S and 5S ribosomal DNA loci as well as centromeric and pericentromeric repeats that organize into chromocenters during interphase. During developmental phase transitions such as seed maturation, germination, seedling growth and flowering that require large-scale reprogramming of gene expression patterns, the organization of repetitive sequences into chromocenters dynamically changes. Here we illustrate recent studies that shed light on the heterochromatin dynamics in cotyledons, the first aerial tissues preformed in the embryo, and in true leaves. We will summarize available data for the 5S rDNA repeat loci, in particular their chromatin organization and expression dynamics during the first days of post-germination development, and discuss how the plant accommodates 5S rRNA transcription during large-scale chromatin reorganization events.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Arabidopsis/crescimento & desenvolvimento , Cotilédone/embriologia , Cotilédone/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação , Heterocromatina/genética , Heterocromatina/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismoRESUMO
Transcription Factor IIIA (TFIIIA) is specifically required for transcription of 5S ribosomal RNA, an essential component of the ribosome. The TFIIIA protein, found in every organism, has been characterized in several species. It shows remarkably poor conservation of primary protein sequence, but all orthologues analyzed carry several C2H2-zinc fingers that are required for TFIIIA binding to both 5S ribosomal DNA (rDNA) and RNA (rRNA). Alignments of TFIIIA protein and 5S rRNA gene sequences suggest a parallel evolution of the transcription factor and its natural binding site, the internal control region of the 5S rRNA gene. We discuss here how TFIIIA expression and availability in the cell is tightly regulated at the transcriptional, post-transcriptional and post-translational level to ensure adequate amounts of TFIIIA protein depending on cell type and developmental stage. This article is part of a Special Issue entitled: Transcription by Odd Pols.
Assuntos
Fator de Transcrição TFIIIA/química , Transcrição Gênica , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Evolução Molecular , Humanos , RNA Ribossômico 5S/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Xenopus , Dedos de ZincoRESUMO
Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA.
Assuntos
Processamento Alternativo , Arabidopsis/genética , RNA Ribossômico 5S/biossíntese , Fator de Transcrição TFIIIA/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Éxons , Regulação da Expressão Gênica de Plantas , Proteólise , RNA de Plantas/genética , Transcrição GênicaRESUMO
The 18S, 5.8S and 25S rRNAs, which result from the 45S precursor, together with 5S rRNAs, are central components of the ribosome. The integration of one molecule of each rRNA per ribosome necessitates an elaborate coordination between transcriptions of the two ribosomal DNA (rDNA) families. Even though 5S rDNA is transcribed by RNA polymerase III and 45S rDNA by RNA polymerase I, the two rDNA families present certain similarities in their transcriptional regulation. This review aims to compare 5S and 45S rRNA genes in the plant model Arabidopsis thaliana in terms of organization, transcription and regulation, and draws parallels between the two rDNA families.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , DNA Polimerase III/metabolismo , DNA Polimerase I/metabolismo , RNA Ribossômico 5S/genética , RNA Ribossômico/genética , Proteínas de Arabidopsis/genética , DNA Polimerase I/genética , DNA Polimerase III/genética , DNA Ribossômico/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genes de RNAr , RNA de Plantas/genética , Transcrição GênicaRESUMO
In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas , Genes de RNAr/genética , Mutação/genética , Fosfoproteínas/metabolismo , RNA de Plantas/genética , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/enzimologia , DNA Espaçador Ribossômico/genética , Perfilação da Expressão Gênica , Histonas/metabolismo , Região Organizadora do Nucléolo/genética , Nucleossomos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Polimerase I/metabolismo , RNA Ribossômico/genética , Sequências Repetitivas de Ácido Nucleico/genética , Transcrição Gênica , NucleolinaRESUMO
The plant-specific RNA polymerases Pol IV and Pol V are essential to RNA-directed DNA methylation (RdDM), which also requires activities from RDR2 (RNA-Dependent RNA Polymerase 2), DCL3 (Dicer-Like 3), AGO4 (Argonaute), and DRM2 (Domains Rearranged Methyltransferase 2). RdDM is dedicated to the methylation of target sequences which include transposable elements, regulatory regions of several protein-coding genes, and 5S rRNA-encoding DNA (rDNA) arrays. In this paper, we have studied the expression of the 5S-210 transcript, a marker of silencing release at 5S RNA genes, to show a differential impact of RNA polymerases IV and V on 5S rDNA arrays during early development of the plant. Using a combination of molecular and cytological assays, we show that Pol IV, RDR2, DRM2, and Pol V, actors of the RdDM, are required to maintain a transcriptional silencing of 5S RNA genes at chromosomes 4 and 5. Moreover, we have shown a derepression associated to chromatin decondensation specific to the 5S array from chromosome 4 and restricted to the Pol V-loss of function. In conclusion, our results highlight a new role for Pol V on 5S rDNA, which is RdDM-independent and comes specifically at chromosome 4, in addition to the RdDM pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Metilação de DNA , DNA de Plantas/metabolismo , DNA Ribossômico/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromossomos de Plantas , RNA Polimerases Dirigidas por DNA/genética , RNA de Plantas/metabolismo , Transcrição GênicaRESUMO
We have investigated the chromatin structure of 5S rDNA, a heterochromatic pericentromeric tandemly repeated family, at 2, 3, 4 and 5 days post-germination. Our results revealed a large-scale reorganization of 5S rDNA chromatin that occurs during the first days of development. Unexpectedly, there is a decondensation followed by a 're'condensation of 5S rDNA chromatin, to obtain almost mature nuclei 5 d post-germination. The reorganization of 5S rDNA chromatin is accompanied by a rapid and active demethylation of 5S rDNA mediated by the ROS1 (repressor of silencing 1) demethylase, whereas the plant-specific RNA polymerase IV (Pol IV) is essential to the 5S chromatin 're'condensation. In conclusion, Pol IV and ROS1 collaborate to unlock the 5S rDNA chromatin inherited from the seed, and establish adult features.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Montagem e Desmontagem da Cromatina , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Ribossômico 5S/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metilação de DNA , DNA de Plantas/genética , DNA Ribossômico/genética , RNA Polimerases Dirigidas por DNA/genética , Germinação , Proteínas Nucleares/genéticaRESUMO
5S ribosomal DNA (5S rDNA) is organized in tandem repeats on chromosomes 3, 4 and 5 in Arabidopsis thaliana. One part of the 5S rDNA is located within the heterochromatic chromocenters, and the other fraction forms loops with euchromatic features that emanate from the chromocenters. We investigated whether the A. thaliana heterochromatin, and particularly the 5S rDNA, is modified when changing the culture conditions (cultivation in growth chamber versus greenhouse). Nuclei from challenged tissues displayed larger total, as well as 5S rDNA, heterochromatic fractions, and the DNA methyltransferase mutants met1 and cmt3 had different impacts in Arabidopsis. The enlarged fraction of heterochromatic 5S rDNA was observed, together with the reversal of the silencing of some 5S rRNA genes known as minor genes. We observed hypermethylation at CATG sites, and a concomitant DNA hypomethylation at CG/CXG sites in 5S rDNA. Our results show that the asymmetrical hypermethylation is correlated with the ageing of the plants, whereas hypomethylation results from the growth chamber/culture conditions. In spite of severely reduced DNA methylation, the met1 mutant revealed no increase in minor 5S rRNA transcripts in these conditions. The increasing proportion of cytosines in asymmetrical contexts during transition from the euchromatic to the heterochromatic state in the 5S rDNA array suggests that 5S rDNA units are differently affected by the (hypo and hyper)methylation patterns along the 5S rDNA locus. This might explain the different behaviour of 5S rDNA subpopulations inside a 5S array in terms of chromatin compaction and expression, i.e. some 5S rRNA genes would become derepressed, whereas others would join the heterochromatic fraction.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Metilação de DNA , Genes de Plantas , RNA Ribossômico 5S/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Inativação Gênica , Genes de Plantas/genética , Sequências de Repetição em Tandem , Fatores de TempoRESUMO
The Arabidopsis thaliana genome comprises around 1,000 copies of 5S rRNA genes encoding both major and minor 5S rRNAs. In mature wild-type leaves, the minor 5S rRNA genes are silent. Using different mutants of DNA methyltransferases (met1, cmt3 and met1 cmt3), components of the RNAi pathway (ago4) or post-translational histone modifier (hda6/sil1), we show that the corresponding proteins are needed to maintain proper methylation patterns at heterochromatic 5S rDNA repeats. Using reverse transcription-PCR and cytological analyses, we report that a decrease of 5S rDNA methylation at CG or CNG sites in these mutants leads to the release of 5S rRNA gene silencing which occurred without detectable changes of the 5S rDNA chromatin structure. In spite of severely reduced DNA methylation, the met1 cmt3 double mutant revealed no increase in minor 5S rRNA transcripts. Furthermore, the release of silencing of minor 5S rDNAs can be achieved without increased formation of euchromatic loops by 5S rDNA, and is independent from the global heterochromatin content. Additionally, fluorescence in situ hybridization with centromeric 180 bp repeats confirmed that these highly repetitive sequences, in spite of their elevated transcriptional activity in the DNA methyltransferase mutants (met1, cmt3 and met1 cmt3), remain within chromocenters of the mutant nuclei.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de RNAr/genética , RNA Ribossômico 5S/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , DNA-Citosina Metilases/genética , DNA-Citosina Metilases/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Hibridização In Situ , Mutação , Plantas Geneticamente Modificadas , Sequências Repetitivas de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Nucleolin is one of the most abundant protein in the nucleolus and is a multifunctional protein involved in different steps of ribosome biogenesis. In contrast to animals and yeast, the genome of the model plant Arabidopsis thaliana encodes two nucleolin-like proteins, AtNUC-L1 and AtNUC-L2. However, only the AtNUC-L1 gene is ubiquitously expressed in normal growth conditions. Disruption of this AtNUC-L1 gene leads to severe plant growth and development defects. AtNUC-L1 is localized in the nucleolus, mainly in the dense fibrillar component. Absence of this protein in Atnuc-L1 plants induces nucleolar disorganization, nucleolus organizer region decondensation, and affects the accumulation levels of pre-rRNA precursors. Remarkably, in Atnuc-L1 plants the AtNUC-L2 gene is activated, suggesting that AtNUC-L2 might rescue, at least partially, the loss of AtNUC-L1. This work is the first description of a higher eukaryotic organism with a disrupted nucleolin-like gene and defines a new role for nucleolin in nucleolus structure and rDNA chromatin organization.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/análise , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Núcleo Celular/química , DNA Ribossômico/metabolismo , Genoma de Planta/genética , Heterocromatina/química , Heterocromatina/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Fosfoproteínas/genética , RNA Ribossômico/biossíntese , Proteínas de Ligação a RNA/análise , NucleolinaRESUMO
The heterochromatic regions around centromeres of animal and plant chromosomes are composed of tandem repetitive sequences, interspersed with transposons and transposon derivatives. These sequences are largely transcriptionally silent and highly methylated, and are associated with specifically modified histones. Although embedded in heterochromatin, Arabidopsis 5S ribosomal RNA genes are among the most highly transcribed genes. However, some 5S genes are silenced, and we show here that this silencing can be suppressed by a reduction in CG methylation. Importantly, we show that mutation of MORPHEUS' MOLECULE 1 (MOM1) releases 5S repeat silencing independently of chromatin properties, as illustrated by the absence of detectable alteration of DNA and histone H3 methylation patterns. MOM1 also prevents transcription of 180-bp satellite repeats and 106B dispersed repeats but not of transposons. Our results provide evidence that transcription of densely methylated and highly repetitive heterochromatic sequences is controlled by two distinct epigenetic silencing pathways, one dependent on and the other independent of DNA methylation.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Proteínas Nucleares/genética , Fatores de Transcrição/genética , ATPases Associadas a Diversas Atividades Celulares , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Elementos de DNA Transponíveis , Mutação , Proteínas Nucleares/metabolismo , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In the Arabidopsis accession Columbia, 5S rDNA is located in the pericentromeric heterochromatin of chromosomes 3, 4, and 5. Both a major and some minor 5S rRNA species are expressed from chromosomes 4 and 5, whereas the genes on chromosome 3 are not transcribed. Here, we show that 5S rDNA methylation is reduced in 2-day-old seedlings versus 4-day-old or older aerial plant tissues, and the minor 5S rRNA species are expressed most abundantly at this stage. Similarly, when 5S rDNA is demethylated by 5-azacytidine treatment or via the decrease in DNA methylation1 (ddm1) mutation, the expression of minor 5S rRNA species is increased. We also show that in leaf nuclei of mature wild-type plants, the transcribed fraction of 5S rDNA forms loops that emanate from chromocenters. These loops, which are enlarged in nuclei of mature ddm1 plants, are enriched for histone H3 acetylated at Lys-9 and methylated at Lys-4 compared with the heterochromatic chromocenters. Up to 4 days after germination, heterochromatin is not fully developed: the 5S rDNA resides in prechromocenters, does not form conspicuous loops, and shows the lowest transcription level. Our results indicate that the expression and chromatin organization of 5S rRNA genes change during heterochromatin establishment.
Assuntos
Arabidopsis/genética , Cromatina/genética , DNA Ribossômico/genética , Heterocromatina/genética , RNA Ribossômico 5S/genética , Transcrição Gênica/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Azacitidina/farmacologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Mutação , RNA Ribossômico 5S/metabolismo , Fatores de Tempo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In vivo, we have already shown that only two of the 5S rDNA array blocks of the Arabidopsis thaliana genome produce the mature 5S rRNAs. Deletions and point mutations were introduced in an Arabidopsis 5S rDNA-transcribed region and its 5'- and 3'-flanks in order to analyse their effects on transcription activity. In vitro transcription revealed different transcription control regions. One control region essential for transcription initiation was identified in the 5'-flanking sequence. The major sequence determinants were a TATA-like motif (-28 to -23), a GC dinucleotide (-12 to -11), a 3-bp AT-rich region (-4 to -2) and a C residue at -1. They are important for both accurate transcription initiation and transcription efficiency. Transcription level was regulated by polymerase III (Pol III) re-initiation rate as in tRNA genes in which TATA-like motif is involved. Active 5S rDNA transcription additionally required an intragenic promoter composed of an A-box, an Intermediate Element (IE) and a C-box. Double-stranded oligonucleotides corresponding to different fragments of the transcribed region, used as competitors, revealed the main importance of internal promoter elements. A stretch of four T is sufficient for transcription termination. Transcription of Arabidopsis 5S rDNA requires 30 bp of 5'-flanking region, a promoter internal to the transcribed region, and a stretch of T for transcription termination.
Assuntos
Arabidopsis/genética , Genes de RNAr/genética , RNA Ribossômico 5S/genética , Transcrição Gênica/genética , Região 3'-Flanqueadora/genética , Região 5'-Flanqueadora/genética , Sequência de Bases , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Dados de Sequência Molecular , Mutação , Deleção de SequênciaRESUMO
Thus far, no transcription factor IIIA (TFIIIA) from higher plants has been cloned and characterized. We have cloned and characterized TFIIIA and ribosomal protein L5 from Arabidopsis thaliana. Primary sequence comparison revealed a high divergence of AtTFIIIA and a relatively high conservation of AtL5 when compared with other organisms. The AtTFIIIA cDNA encodes a protein with nine Cys(2)-His(2)-type zinc fingers, a 23 amino acid spacer between fingers 1 and 2, a 66 amino acid spacer between fingers 4 and 5, and a 50 amino acid non-finger C-terminal tail. Aside from the amino acids required for proper zinc finger folding, AtTFIIIA is highly divergent from other known TFIIIAs. AtTFIIIA can bind 5S rDNA, as well as 5S rRNA, and efficiently stimulates the transcription of an Arabidopsis 5S rRNA gene in vitro. AtL5 identity was confirmed by demonstrating that this protein binds to 5S rRNA but not to 5S rDNA. Protoplast transient expression assays with green fluorescent protein fusion proteins revealed that AtTFIIIA is absent from the cytoplasm and concentrated at several nuclear foci including the nucleolus. AtL5 protein accumulates in the nucleus, especially in the nucleolus, and is also present in the cytoplasm.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Ribossômicas/genética , Fator de Transcrição TFIIA/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Pegada de DNA/métodos , DNA Complementar/química , DNA Complementar/genética , DNA Ribossômico/metabolismo , Desoxirribonuclease I/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fator de Transcrição TFIIA/metabolismo , Transcrição GênicaRESUMO
Cytosine methylation was studied at the level of the euchromatin/heterochromatin transition genomic region of the Arabidopsis chromosome 5 left arm. It has been shown using a monoclonal antibody against 5-methylcytosines that the density of DNA methylation increases from the euchromatin towards the heterochromatin. YACs mapped along this region were characterized for their repeated sequences content. Some of them, corresponding to euchromatin, euchromatin/heterochromatin border and heterochromatin regions, were used as probes for a Southern blot analysis of methylation. This revealed that the degree of mCmCGG and GATmC methylation increases significantly from the euchromatin towards the heterochromatin. Moreover, an analysis of cytosine methylation levels (% of 5-methylcytosine) of different DNA fragments, inside the same genomic region, was performed using PCR and/or Southern blot approaches. There is a gradual increase of methylation along the genomic region analyzed: CpG methylation in the euchromatic fraction, CpG and CpNpG methylation at the euchromatin/heterochromatin transition and an additional asymmetrical methylation in the repeated-heterochromatic fraction. The most methylated repeated family at CpG, CpNpG and asymmetrical sites is the 5S ribosomal DNA, highly methylated even though it is transcribed.
Assuntos
Arabidopsis/genética , Cromossomos/genética , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Eucromatina/metabolismo , Genoma de Planta , Heterocromatina/metabolismo , 5-Metilcitosina , Southern Blotting , Cromossomos Artificiais de Levedura/genética , Ilhas de CpG , DNA de Plantas/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Regulação da Expressão Gênica de Plantas , Hibridização in Situ Fluorescente , Mutação , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiaeRESUMO
Methylation has often been correlated with transcriptional inhibition of genes transcribed by polymerase II, but its role on polymerase III genes is less well understood. Using the genomic sequencing technique, we have analysed the methylation pattern of the different 5S-rDNA arrays of the Arabidopsis genome. Every cytosine position within the 5S sequence is highly methylated whatever the context - CpG, CpNpG or non-symmetrical. The methylation pattern of both transcribed and non-transcribed 5S units is similar, with no preferential methylated or unmethylated site. These results, taken together with 5-azacytidine treatments and in vitro transcription experiments using methylated 5S templates, demonstrate that 5S rRNA gene transcription is not inhibited by methylation. Non-transcribed 5S arrays are more subject to transition mutations resulting from deamination of 5-methylcytosines, leading to CpG depletions and an increasing A + T content. As there were no detectable differences in methylation, this implies more efficient repair and/or selection pressure in transcribed 5S-blocks.
Assuntos
Arabidopsis/genética , Metilação de DNA , RNA Ribossômico 5S/genética , Azacitidina/farmacologia , Citosina/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação , RNA Ribossômico 5S/metabolismo , Nicotiana/genética , Fatores de Transcrição/fisiologiaRESUMO
One major 5S RNA, 120 bases long, was revealed by an analysis of mature 5S RNA from tissues, developmental stages, and polysomes in Arabidopsis thaliana. Minor 5S RNA were also found, varying from the major one by one or two base substitutions; 5S rDNA units from each 5S array of the Arabidopsis genome were isolated by PCR using CIC yeast artificial chromosomes (YACs) mapped on the different loci. By using a comparison of the 5S DNA and RNA sequences, we could show that both major and minor 5S transcripts come from only two of the genomic 5S loci: chromosome 4 and chromosome 5 major block. Other 5S loci are either not transcribed or produce rapidly degraded 5S transcripts. Analysis of the 5'- and 3'-DNA flanking sequence has permitted the definition of specific signatures for each 5S rDNA array.