DNA methylation is essential for mammalian development and transcriptional repression of genes and retrotransposons during embryo development and in somatic cells. The patterns of DNA methylation are established by de novo DNA methyltransferases, which are regulated by developmental signalling and require access to chromatin. Besides DNA methyltransferases, other proteins have recently been implicated in DNA methylation, such as the ATP-dependent chromatin remodeler LSH. The absence of LSH in mouse embryos leads to defects in DNA methylation and development. In relation to this, mutations in LSH have been found to cause Immunodeficiency-Centromeric instability-Facial anomalies (ICF) syndrome. This syndrome is characterized by centromeric instability and CpG hypomethylation of centromeric satellite repeats, and is most often caused by mutations in the catalytic domain of the DNA methyltransferase DNMT3B. LSH is essential for developmentally programmed de novo DNA methylation of large chromosomal domains including promoters of protein coding genes and repetitive sequences. Importantly, fibroblasts derived from chromatin remodeling ATPase LSH-null mouse embryos, which lack DNA methylation at transposons and specific gene promoters, are capable of re-establishing normal patterns of DNA methylation and transcriptional silencing of misregulated genes upon re-expression of LSH. The ATP hydrolysis by LSH is essential for its function in gene silencing and de novo DNA methylation. However, the molecular mechanisms of LSH-dependent gene silencing and de novo DNA methylation are yet unclear. Here we use an inducible system that enables controlled expression of LSH in Lsh-null mouse embryonic fibroblasts (MEFs) to follow chromatin dynamics, transcriptional silencing and establishment of de novo DNA methylation. This conditionally reversible Lsh knockout cellular system allowed us to study the order of events occurring immediately after LSH restoration in MEF cell lines in order to elucidate the molecular mechanism of LSH-dependent gene silencing. We have demonstrated that LSH upon its restoration localises to the promoters of LSH-dependent loci leading to a mild decrease in the occupancy of H3, which reinforces the previously shown role of LSH as a chromatin remodeler. Simultaneously, there is removal of acetyl groups from H3 tails when LSH is bound to these target regions, which might be facilitated by the interaction of HDACs with LSH. The removal of H3Ac marks is followed by deposition of H3K9me2 by G9a/GLP histone methylases at the same time point when misregulated genes are silenced. This suggests that LSH creates a suitable substrate for G9a/GLP promoting gene silencing. Surprisingly, transcriptional repression occurs without acquisition of DNA methylation at the promoters of these loci. This order of events implies that LSH plays a role as a chromatin remodeler leading to changes in chromatin structure and modifications that facilitate epigenetic gene silencing without DNA methylation in the initial period when LSH is restored in MEF cell lines. Furthermore, deposition of H3K9me2 by the G9a/GLP complex is critical for silencing of specific genes, but not for repetitive elements such as IAPs. The histone modification H3K27me3 seems to play a transitory role in the silencing of IAP retrotransposons in the absence of G9a/GLP activity. In conclusion, this work has demonstrated that changes in chromatin modifications leading to a transcriptionally repressive chromatin state can be established in somatic cells by the chromatin remodeler LSH without acquisition of DNA methylation. This suggests that the primary role of LSH is to promote changes in chromatin structure and modifications that lead to gene silencing and not DNA methylation, which most likely occurs as a consequence of transcriptional silencing.

The rapid postharvest deterioration of the roots, known as postharvest physiological deterioration (PPD), has been a major problem to the utilisation and development of cassava (Manihot esculenta Crantz) as a food and industrial crop. PPD usually occurs within two to three days after harvest and it is characterised by a blue-black discoloration of the roots, which renders the roots unpalatable and unmarketable. Scopoletin, which is synthesised de novo and released from its glucoside, scopolin, during PPD, plays a central role in this discoloration response. Interconversion of scopoletin and scopolin, which is catalysed by scopoletin-glucosyltransferase (scopoletin-GT) and scopolin-beta-glucosidase (scopolin-BG), regulates homeostasis of scopoletin in the cells. However, how this interconversion contributes to root discoloration development is poorly understood. In the present study, we identified and characterised cassava genes for the enzymes that are responsible for the interconversion of scopoletin and scopolin, subsequently manipulated their expression in transgenic cassava through scopoletin-GT RNAi gene silencing and scopolin-BG overexpression constructs. These approaches would potentially alter scopoletin and scopolin content in the root, thereby affecting PPD response. A BLAST search for homologous cassava genes revealed that scopoletin-GT and scopolin-BG are encoded by multiple genes, most of which belong to glucosyltransferase family-1 (GT1) and glycosyl hydrolase family-1 (GH1), respectively. Scopoletin-GT-down-regulated and scopolin-BG overexpressed transgenic cassava lines showed reduced not only scopolin but surprisingly also scopoletin, and delayed PPD. Additionally, other coumarins esculetin and esculin were also identified and both scopoletin-GT, MeSGT1, and scopolin-BG, BGLU23, were up-regulated during PPD development at day 4 and day 2, respectively. Our study reveals that disrupting the interconversion of scopoletin and scopolin by inhibiting scopoletin-GT and overexpressing scopolin-BG led to the decrease of both scopoletin and scopolin content and delayed PPD in cassava. These findings provide useful insights into the role of interconversion of scopoletin and scopolin in cassava PPD response and may suggest alternative ways to tackle PPD.

O HLB tem sido uma das principais doenças responsáveis pelos prejuízos econômicos enfrentados na citricultura mundial. Essa doença é associada a três espécies de bactérias de colonização restrita ao floema das plantas, as quais são transmitidas pelo psilídeo Diaphorina citri. Uma vez que na?o ha? medidas curativas para a doenc?a, o manejo baseia-se em medidas preventivas incluindo o controle do inseto vetor. O silenciamento gênico por interferência de RNA (RNAi) é uma nova ferramenta biotecnológica no controle de pragas, pois, proporciona uma ação eficiente e gene-espécie específica. Entretanto, essa tecnologia depende de análises de expressão gênica com genes de referência adequados e estáveis. Este trabalho buscou selecionar e validar a estabilidade da expressão de seis genes de referência potenciais de D. citri sob diferentes condições experimentais e avaliar a biologia deste inseto em plantas transgênicas de laranja doce expressando dsRNA do gene DcV-ATPase-A visando o seu controle. Para isso, foram avaliados: 1) a estabilidade de seis genes candidatos a referência: fator de elongação 1alfa (EF 1-α), actina (ACT), gliceradeído-3-fosfato desidrogenase (GAPDH), proteina ribossomal L7 (RPL7), proteina ribossomal L17 (RPL17) e alfa tubulina (TUB), através de diferentes algorítimos matemáticos: Delta Ct, NormFinder, BestKeeper e Genorm em cinco estádios de desenvolvimento (Ninfas de 1° e 2° ínstar; Ninfa de 3° ínstar; Ninfas de 4° e 5° ínstar , Adulto de 1 dia; Adulto de 10 dias) e dois hospedeiros (Citrus sinensis e Murraya paniculata). O ranquamento final dos genes foi obtido através do RefFinder e a validação dos mesmos através do gene V-ATPase-A; 2) a sobrevivência de psilídeos adultos alimentados em plantas transgênicas; a biologia da D. citri em plantas transgênicas expressando o dsRNA do gene DcV-ATPase-A; e a expressão relativa deste gene. Recomenda-se a utilização de dois genes de referência quando consideram-se diferentes hospedeiros e fases de desenvolvimento distintas. Para análise de expressão gênica, independentemente da fase de desenvolvimento do inseto, quando utiliza-se M. paniculata como hospedeiro de criação, recomenda-se a utilização dos genes GAPDH e RPL7, enquanto que para C. sinensis sugerem-se os genes GAPDH e EF 1-α. Os bioensaios demonstraram a internalização pelo inseto das moléculas de dsRNA/siRNA produzidas pelas plantas transgênicas e o potencial de redução da expressão do gene alvo pelo RNAi. Apesar da ausência de fenótipo letal em psilídeos adultos alimentados nas plantas transgênicas, alterações significativas na duração, sobrevivência e longevidade foram verificadas em D. citri quando esta desenvolveu seu ciclo biológico nas plantas que expressam dsRNA do gene DcVAPTase-A. / HLB has been one of the main diseases responsible for the economic losses faced in the citriculture worldwide. This disease is associated to three species of colonization bacteria restricted to the phloem of the plants, which are transmitted by the psyllid Diaphorina citri. Since there are no curative methos for the disease, management is based on preventive methods including vector insect control. Gene silencing by RNA interference (RNAi) has been presented as a new biotechnological tool in pest control, since it provides a efficient and especific gene-specie control. However, this technology depends on analyzes of gene expression with suitable and stable reference genes. Thus, this work aimed to select and validate the stability of the expression of six potential reference genes of D. citri under different experimental conditions and to evaluate the biology of this insect on sweet orange transgenic plants expressing dsRNA of DcV-ATPase-A gene aiming their control. In order to do this, it was evaluated: 1) the stability of six candidate reference genes: elongation factor 1 alpha (EF 1-α), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L7 (RPL7), ribosomal protein L17 (RPL17) e alpha tubulin (TUB), through different mathematical algorithms: Delta Ct, NormFinder, BestKeeper and Genorm in five developmental stages and two hosts (Citrus sinensis and Murraya paniculata). The final rank was obtained through the RefFinder and validated by the V-ATPase-A gene; 2) the survival of adult psyllids fed on transgenic plants; the biology of D. citri in transgenic plants expressing dsRNA of the DcV-ATPase-A gene; and the relative expression of this gene. According to our results, two reference genes are recommended when different hosts and different developmental stages are considered. For gene expression analysis, regardless the insect developmental stage, when using M. paniculata as a breeding host, it is recommended to use GAPDH and RPL7 genes, whereas for C. sinensis GAPDH and EF 1-α are indicated. The bioassays demonstrated the insect\'s internalization of the dsRNA / siRNA molecules produced by the transgenic plants and the potential for reducing the target gene expression by RNAi. Despite the absence of lethal phenotype in adult psyllids fed to transgenic plants, significant changes in duration, survival and longevity were observed for D. citri when it developed its biological cycle in plants that express dsRNA of the DcVAPTase-A gene.

O vírus da tristeza dos citros (CTV) é uma das maiores ameaças à citricultura mundial. No Brasil, mesmo com a pré-imunização e com a substituição de porta-enxertos, estirpes fortes de CTV ainda causam prejuízos consideráveis. Com o aparecimento da Morte Súbita dos Citros em 1999 e a possível relação desta doença com o CTV, este vírus voltou a figurar como patógeno de importância no cenário da citricultura brasileira. Uma das possíveis soluções para o controle de viroses em fruteiras é a obtenção de plantas transgênicas resistentes ou imunes. O objetivo deste trabalho foi avaliar a resistência ao CTV de plantas transgênicas de laranja \'Hamlin\' contendo três construções gênicas oriundas de seqüências do genoma do CTV. Estas construções gênicas visaram ativar rotas de RNAi (hairpin da capa protéica e seqüência conservada antisenso do CTV) e mecanismos de defesa relacionados à expressão da capa protéica do CTV. As plantas transgênicas foram desafiadas com uma estirpe fraca de CTV (CTV-IAC) por meio de borbulhas e pulgões pretos (Toxoptera citricida Kirkaldy) contendo o vírus. A avaliação da resistênica à replicação viral foi feita por análises de ELISA. As plantas transgênicas foram consideradas não resistentes à infecção e translocação viral quando inoculadas com borbulhas. Entretanto algumas plantas mostraram retardamento da infecção. Não foi possível determinar se houve resistência à transmissão de CTV por pulgões já que a técnica utilizada não foi capaz de infectar os controles de maneira uniforme. / The Citrus tristeza virus (CTV) is one of the greatest threats to the citrus industry worldwide. In Brazil, CTV continues to cause damage through strong strains despite the use of techniques like cross-protection and substitution of intolerant rootstocks. With the appearance and spread of the Citrus Sudden Death disease in 1999 and its possible relation to CTV, this virus was again among important pathogens within the Brazilian citrus industry. One of the possible solutions for controlling virus diseases in fruit crops is the development of immune or resistant transgenic plants. The objective of this work was to evaluate the resistance to CTV of transgenic \'Hamlin\' sweet orange plants containing three transgenic constructs obtained from CTV genomic sequences. The genetic constructs used aimed to activate RNAi defense routes (coat protein hairpin and a conserved sequence from CTV) and resistance mechanisms related to the coat protein expression. The transgenic plants were challenged with a weak strain of CTV, CTV-IAC, by bud and aphid (Toxoptera citricida Kirkaldy) inoculation. The evaluation of viral replication was done by ELISA analysis. The transgenic plants were considered susceptible to viral replication and translocation when bud inoculated. However, a few plants showed retardation of infection. It was not possible to determine resistance in the aphid transmission assay since the controls were not uniformly inoculated.

Psoroptic mange, caused by the Astigmatid mite Psoroptes ovis, is an ectoparasitic disease of significant economic importance to agriculture on a global scale and poses a serious welfare concern. With the current chemotherapeutic controls considered unsustainable, there is pressing need for novel control strategies. RNA interference has been proposed as a potential high throughput approach for the identification of novel therapeutic targets with high specificity, speed and at a relatively low cost compared to the existing methods. The presence of the components of the RNA interference (RNAi) pathway in P. ovis was first confirmed through in silico analyses of the P. ovis transcriptome and, following development of a non-invasive immersion method of double stranded RNA (dsRNA) delivery, gene silencing by RNAi was demonstrated in P. ovis. Statistically-significant reduction of transcript level was measured for the three genes targeted: P. ovis mite group 2 allergen (Pso o 2), P. ovis mu class glutathione S-transferase (PoGST-mu1) and P. ovis beta tubulin (Poβtub). This is the first demonstration of gene silencing by RNAi in P. ovis and provides a key mechanism for mining transcriptomic and genomic datasets in the future for novel targets of intervention against P. ovis. The first assessment of gene silencing was also performed in two related Astigmatid mites of high medical importance; the European house dust mite Dermatophagoides pteronyssinus and the scabies mite Sarcoptes scabiei. A statistically-significant reduction in expression of a D. pteronyssinus mu class glutathione S-transferase (DpGST-mu1) transcript was observed. No significant reduction in expression of a S. scabiei mu class glutathione S-transferase (SsGST-mu1) transcript was observed. Additionally, microRNAs (miRNAs) from the related miRNA pathway were identified in a P. ovis small RNA sample and were sequenced and annotated.

Les miARN sont de petits ARN non codant dont la taille varie entre 21-24 nucléotides. Ils jouent un rôle de régulateurs post-transcriptionnels en utilisant leur complémentarité de séquence avec l’ARN messager (ARNm) cible afin d’induire sa répression. Grâce à la protéine Argonaute 2 (Ago2) dans laquelle les miARN sont incorporés formant ainsi le complexe miRISC, des cofacteurs sont recrutés afin d’induire la dégradation ou le blocage de la traduction de l’ARNm cible. Initialement connus pour réguler leurs cibles dans le cytoplasme, les miARN sont de plus en plus décrits comme étant des régulateurs de l’expression génique au niveau nucléaire. Dans ce travail, nous avons démontré la présence, au sein du noyau, d’un nouveau mécanisme de régulation post-transcriptionel par les miARN dont les facteurs majeurs sont Sfpq et Pspc1 / Micro-RNA, nuclear regulation, gene silencing, SfpqThere is a growing body of evidence about the presence and the activity of the miRISC in the nucleus of mammalian cells. Here we show by quantitative proteomic analysis that Ago2 interacts with the complex formed by Sfpq, Pspc1 and NonO in a RNA-dependent fashion. Sfpq mediates the interaction between miRISC with Pspc1 and NonO in the nucleoplasm. By HITS-CLIP coupled with transcriptomic analysis, we demonstrated that Sfpq specifically controls the downregulation of a subset of crucial let-7a-target mRNAs in stem cells, including Lin28a, Prtg, and Igf2bp1. Sfpq directly binds to specific sequence in the 3'UTR to promote the recruitment of selected nucleoplasmic miRNAs and triggers the decay, as we show for Lin28a mRNA. These results extend the miRNA-mediated post-transcriptional gene silencing into the nucleus and indicate that a dual strategy

Le silencing transcriptionnel limite la transcription des gènes et des éléments transposables dont l’expression pourrait être délétère à la cellule. Il dépend d’une diversité de modifications de la chromatine comme la méthylation ADN ou les marques répressives des histones. De façon à mieux comprendre les mécanismes moléculaires à l’origine du silencing transcriptionnel, nous avons mené une approche de génétique directe à l’aide d’un transgène soumis au silencing dans la plante modèle Arabidopsis thaliana. Cette stratégie nous a permis d'isoler à la fois des mutants déficients pour le maintien du silencing transcriptionnel et des mutations qui empêchent la réactivation transcriptionnelle des éléments transposables en réponse à un stress thermique. Nous avons caractérisé les défauts provoqués par ces mutations en combinant des approches de biologie moléculaire, de cytologie et de génomique.Nous montrons ainsi que MED14, la sous-unité centrale du complexe Mediator, et UVH6, composant du complexe TFIIH, sont requis pour la transcription de l'hétérochromatine en stress thermique. MED14 stimule aussi la transcription de l'hétérochromatine en l'absence de stress, mais ne semble fonctionner qu'en présence de la méthylation ADN. En plus de cette fonction originale, nous identifions un nouveau rôle de MED14 dans le maintien de la méthylation ADN, possiblement via la voie de méthylation ADN dirigée par les petits ARN.Par ailleurs, nos résultats nous ont permis d’identifier le rôle des protéines MAIN et MAIL1, qui définissent une voie de silencing transcriptionnelle indépendante des voies connues jusqu'alors. De façon intéressante, MAIN et MAIL1 possèdent un domaine protéique partagé avec les éléments transposables, qui aurait successivement été capturé par les éléments transposables et leur hôte au cours de l’histoire évolutive des plantes à fleurs.Enfin, en isolant une nouvelle mutation du gène POL2A, nous confirmons le rôle de l’ADN polymérase epsilon dans le silencing transcriptionnel et caractérisons les propriétés chromatiniennes qui dépendent de POL2A. Nous montrons que les défauts de silencing des mutants pol2a corrèlent avec une désorganisation importante de l’hétérochromatine sans diminution drastique des marques qui y sont associées. Au contraire, nous détectons une hyperméthylation ADN prononcée dans le mutant, et explorons différentes hypothèses pour expliquer ce phénotype particulier. Nos données suggèrent que plusieurs mécanismes moléculaires sont à l’origine des défauts des mutants pol2a. Elles confirment le rôle prépondérant de la chromométhylase CMT3 dans la régulation de la méthylation ADN, et suggèrent qu’un stress réplicatif pourrait causer une hyperméthylation de l’ADN.Dans l’ensemble, ces travaux de thèse proposent des pistes de travail dont l’exploration pourrait permettre d’expliquer les effets des déficiences réplicatives dans le maintien du silencing transcriptionnel et de l’homéostasie de la méthylation ADN. Ils suggèrent en outre que MED14 a une fonction dédiée à la transcription de l’hétérochromatine qui pourrait stimuler le maintien de la méthylation ADN. / Transcriptional gene silencing hinders deleterious transcription of some genes and transposable elements. Silencing is maintained by numerous chromatin modifications such as DNA methylation and repressive histone marks. To better understand the molecular mechanisms of silencing, we conducted a forward genetic screen using a transgene reporter system targeted by transcriptional gene silencing in the model plant Arabidopsis thaliana. We isolated a first type of mutants with diminished maintenance of silencing and a second category that displayed deficient release of transgene silencing upon heat stress. We then combined molecular, cytological and genomic methods to characterize the defects associated with these mutations.First, we show that the Mediator subunit MED14 and the TFIIH complex subunit UVH6 are required for heat-stress-induced release of silencing. We further show that MED14, but not UVH6, promotes transcriptional activation of transposable elements in mutant contexts where silencing is defective. Importantly, MED14 is only required when DNA methylation is not affected, suggesting that MED14 has a specialized function to promote transcription of heterochromatin. Furthermore, we show that MED14 promote DNA methylation at targets regulated by RNA-directed DNA methylation.Characterizing mutants from the first category, we unveil the contribution of the MAIN and MAIL1 proteins into transcriptional gene silencing, and show that they likely act through a pathway independent of known silencing factors. Interestingly, MAIN and MAIL1 bear a protein domain that is shared with transposable elements, and that has been captured by transposable elements and genes throughout the evolutionary history of flower plants.Additionally, we confirm the involvement of the DNA polymerase epsilon in transcriptional gene silencing by isolating a new mutation of the POL2A gene among mutants of the first category. We characterize the effects of the pol2a mutation on several heterochromatin properties, and show that the pol2a mutant retains high levels of heterochromatin marks despite having highly disorganized heterochromatin. We actually detect a strong elevation of DNA methylation in the pol2a mutant and explore different hypothesis to explain this unusual phenotype. We show that increased expression of the CMT3 chromomethylase is a likely cause, but that additional molecular mechanisms are probably involved. Further exploration suggests that constitutive replicative stress occurring in pol2a mutants could be an additional cause of DNA hypermethylation.To summarize, this work provide putative causes for DNA hypermethylation and silencing defects in a situation of replicative deficiency. Further investigation will be required to identify the molecular components involved in the mechanism. Our data further suggest that MED14 has a function dedicated to heterochromatin transcription that could promote DNA methylation maintenance.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 90-99).

Variability of transgene expression levels resulting from gene silencing is considered as ahindrance to the successful application of plant genetic engineering. Towards alleviatinggene silencing, I decided to screen for novel genes involved in transgene silencing and toinvestigate how these genes regulate plant development. Genes encoding putative chromatinremodeling factors, especially those including a SET domain, were selected as candidatetargets. A bioinformatic analysis of the Arabidopsis SET genes (AtSET) was performed andthese genes were classified into 6 groups based on the domain architecture. RNA interference (RNAi) vectors were constructed for ~ 20 AtSET genes and wereintroduced into both wild type lines and transgenic lines silenced for a GFP reporter gene.Surprisingly, altered developmental phenotypes were only observed for three constructs,raising questions as to the effectiveness of the RNAi approach for the chosen Arabidopsissystem. To assess this situation, I targeted a phytoene desaturase (PDS) gene using the sameRNAi approach. Inactivation of PDS renders plant a readily identifiable phenotype. Whereasthe RNAi penetrance in Arabidopsis can be very high, the expressivity of RNAi in varioustissues and among different plants can vary dramatically. Contradictory to previous reports,I found that there is correlation between transcript level and silencing phenotype. Possiblereasons for this discrepancy are discussed. No apparent correlation between transgene copynumber and RNAi phenotypes was observed. Among the three RNAi constructs that caused an abnormal development inArabidopsis, K-23 which targets SuvR3 has the highest expressivity and could reactivate asilenced GFP locus. SuvR3 RNAi lines were selfed for six generations and were screenedfor morphological phenotypes. Abnormal number of flower organs, loss of viability of malegametophytes, and decreased seedling germination percentage were found in SuvR3 RNAilines. A progressive increase in both severity and frequency of abnormal phenotypes wereseen in subsequent generations, suggesting an epigenetic regulatory mechanism involvedwith SuvR3. Alternative splicing of SuvR3 was also observed in most of Arabidopsis tissues.One of the protein isoforms, SuvR3, lacks 16 amino acids within the highly conserved SETdomain. Possible effects of isoform interaction are proposed.