RNA interference represents a useful tool for modulating expression of sarcosine metabolism genes and for studying the role of sarcosine in prostate cancer. The thesis "Modulation of sarcosine metabolism by RNA interference" summarizes current state-of-the-art of possible regulation of gene expression using various types of nucleic acids. Furthermore the thesis also deals with the issue of the transfer of these regulatory agents to target tissues and finally it describes sarcosine including its involvement in the metabolic cycles of the cell. The main aim of the experimental part of this work was to determine the influence of knock down sarcosine dehydrogenase (SARDH) on other enzymes involved in sarcosine metabolism. This effect was assessed by determining the gene expression of individual genes encoding the four major sarcosine pathway enzymes by quantitative real-time PCR analysis. Experiments were performed on three different prostatic cell line types, PNT1A, DU-145 and PC3. The most significant differences on level of gene expression were observed in carcinoma cells DU-145 in which a significant increase in gene expression of dimethylglycine dehydrogenase (DMGHD) and sarcosine oxidase (PIPOX) was observed after applications of siRNA targeting the SARDH.

No description available.

Cell wall is the major barrier in the delivery of biomolecules such as nucleic acids into the plant cell. Biological (bacteria or viruses) and biolistic (particle-based) methods are used to deliver nucleic acids into the plant cell. However, these methods have significant limitations when it comes to species range, scalability, and field assays. In this work, we report the use of layered double hydroxide (LDH) topically applied to deliver RNA molecules into the plant cell. LDH were assembled by methanol-based co-precipitation of magnesium and aluminum nitrate solution with sodium hydroxide and finally dispersed in deionized water. The assembled LDH were physically characterized by AFM, zeta-sizer and their binding to RNA was confirmed by gel electrophoresis. LDH complexed with double stranded RNA (dsRNA) was topically applied to Nicotiana benthamiana leaves. As a model system, virus specific dsRNA-LDH complexes were used to activate cellular RNAi machinery against Tomato Yellow leaf Curl Virus (TYLCV) in N. benthamiana plants. Our results demonstrated that topical application of the TYLCV specific dsRNA-LDH complexes reduce viral genome accumulation and viral symptoms development. Similarly, dsRNA-LDH protected plants produce typical leaves, flowers, and seeds, confirming efficient virus resistance compared unprotected TYLCV infected plants. Topical application and noninvasive delivery of nucleic acid has several advantages, as these methods are specie independent, easy to scale up, applied with low-pressure spray, requires no tissue culture and no sophisticated equipment. The LDH based noninvasive delivery of nucleic acids has the capability to overcome the cell wall barrier limitations and will open new opportunities to exploit the full potential of cellular machinery to produce resilient plants and insure sustainable food production.

No description available.

This work provides a comparative and genetic analysis of the responses to water stress in desiccation-tolerant and desiccation-sensitive nematodes. Caenorhabditis elegans, a model organism for the study of development, aging, and cell biology was shown to be a desiccation-sensitive organism that survives relative humidities above 40\% for periods of up to seven days. Transcripts from the desiccation-tolerant species Panagrolaimus rigidus AF36 and sp. PS1579, which were expressed uniquely during separate desiccation and osmotic stresses, as well as during recovery from exposure to the dual stresses, were cloned. These sequences were used to search for similarities in the genome sequence data of C. elegans. Putative anhydrobiotic-related transcripts were identified that potentially encode heat shock protein 70, late embryogenic abundant protein, and trehalose-phosphate synthase. Other putative genes that were identified within eight separate libraries encode proteins involved in transcription (histones), protein biosynthesis (ribosomal proteins, elongation factors), protein degradation (ubiquitin, proteases), and transport and cell structure (actin, collagen). Gene ontology analysis of the cloned transcripts revealed that developmental processes are activated during exposure to the stresses as well as during recovery, which may suggest a "rejuvenation" process as a key to survival in Panagrolaimus nematodes. Genes that were up-regulated during desiccation stress in C. elegans were classified as belonging either to an early response (until 12 hours of stress), or to a late response (after 12 hours of stress). The early response was characterized by the up-regulation of a large number of genes encoding mono-oxygenases, which may suggest onset of oxidation stress during desiccation of C. elegans. The late response was characterized by the appearance of transcripts encoding proteins of the immune system, heat shock proteins (protein denaturation), and superoxide dismutases (oxidation damage). Genes in C. elegans that were down-regulated in response to desiccation stress include those encoding proteases and lysozymes (metabolic shutdown). Genes that encode channel proteins (water homeostasis) were found among the transcripts up-regulated during recovery of C. elegans. The up-regulation of gpdh-1 and hmit-1.1, two transcripts linked to hyperosmotic stress, suggest that osmotic stress is experienced by C. elegans. Comparison of these data with those obtained from exposure of C. elegans to a range of other stresses showing that the nematode C. elegans uses specific transcripts for the desiccation response; transcripts that are not induced in other stresses such as heat, anoxia or starvation. In addition, transcripts regulated during desiccation stress of C. elegans were also regulated during dauer formation, which may indicate common stress tolerant mechanisms. Recent studies in mammalian cells and C. elegans have shown that microRNAs are able to degrade and to sequester mRNA especially during stress in so called stress bodies. In this study, C. elegans microRNA knock-outs showed a significant decrease in desiccation stress survival compared to wild type C. elegans which may suggest the importance of microRNAs for stress survival in C. elegans and other organisms. / Ph. D.

Amino acids are assimilated by membrane-associated transporters into and out of enterocytes either in their free form or in the form of peptides. The peptide transporter, PepT1, is thought to be the major facilitator of peptide transport in the enterocyte. It is unknown if the peptide transporters and free amino acid transporters operate in a compensatory fashion to regulate the amino acid balance within the enterocyte. Therefore, the objective was to examine the regulatory balance between PepT1 and other peptide and free amino acid transporters in enterocytes. The Mouse Small Intestinal Epithelial (MSIE) cells are conditionally immortalized. It was found that MSIE cells express BoAT1, CAT1, CAT2, LAT1, y+LAT1, and y+LAT2, but not PepT1, EAAT3, Bo,+AT, or LAT2, making this model similar to the basolateral membrane of enterocytes. Growing MSIE cells at high temperatures did not affect the nutrient transporter gene expression profile of these cells. Thus, the human colon carcinoma (Caco-2) cell line was used as a small intestinal in vitro model for this study. These cells express PepT1, HPT1, PTR3 EAAT1, EAAT3, rBAT, Bo,+AT CAT1, LAT1, y+LAT1, y+LAT2, ABCC3, ABCC4, which increased from D0 to D21 post confluency, indicating cell maturation. In Caco-2 cells, PepT1 gene silencing was induced in Caco-2 cells. Despite an reduction of PepT1 gene (82%, P < 0.05) protein (96%), no significant difference in any peptide (HPT1, PTR3, ABCC3, ABCC4) or free amino acid transporters (EAAT1, EAAT3, rBAT, Bo,+AT, BoAT1, CAT1, CAT2, LAT1, LAT2, y+LAT1, y+LAT2) between Caco-2 cells treated with PepT1 siRNA and Caco-2 cells treated with Control siRNA was observed. These results suggest no compensation at the gene expression level of these transporters in response to a reduction of PepT1. To account for the limitations of an in vitro and PepT1 kockout mouse model, transgenic chicken models were pursued. Potential cPepT1 overexpressing, cPepT1 shRNA or control shRNA expressing G0 chickens were generated by embryo injection of pseudolentiviral particles followed by ex ovo egg culture. Overall, 9 potential G0 cPepT1 overexpressing chickens, 15 potential G0 cPepT1 shRNA expressing chickens, and 4 potential G0 control shRNA expressing chickens were generated. / Ph. D.

Chez la majorité des eucaryotes, les petits ARN (miRNA et siRNA) jouent des rôles essentiels au cours du développement, dans les réponses adaptatives aux stress, et dans la maintenance de la stabilité génétique. Les plantes codent quatre enzymes RNaseIII de type DICER-LIKE (DCL). DCL1, produit les miRNAs, tandis que DCL2, DCL3 et DCL4 produisent des siRNAs des tailles diverses. Les plantes codent également des enzymes appelées RNASE-THREE-LIKE (RTL) auxquelles il manque certains domaines spécifiques aux DCLs, et dont la fonction est largement inconnue.Des plantes sur-exprimant RTL1 montrent des défauts morphologiques, et n'accumulent pas les siRNAs produits par DCL2, DCL3 ou DCL4, indiquant que RTL1 est un suppresseur général des voies de siRNA chez les plantes. L’activité de RTL1 nécessite un domaine RNaseIII fonctionnel. RTL1 ne s'exprime naturellement que faiblement dans les racines, mais l'infection virale induite fortement son expression dans les feuilles, ce qui suggère que l’induction de RTL1 est une stratégie générale utilisée par les virus pour contrer la défense antivirale basée sur siRNAs. En accord avec cette hypothèse, les plantes transgéniques sur-exprimant RTL1 sont plus sensibles à l'infection par le TYMV que des plantes de type sauvage, probablement parce que RTL1 empêche la production des siRNAs dirigés contre les RNA viraux. Cependant, les plantes transgéniques sur-exprimant RTL1 ne sont pas plus sensibles à l'infection par le TCV, TVCV ou le CMV, qui codent les suppresseurs de RNA silencing (VSR) plus puissants que le TYMV. En effet, le VSR de TCV inhibe l'activité de RTL1, suggérant que l'induction de l’expression de RTL1 par les virus et l’amortissement de l’activité de RTL1 par leurs VSRs est une double stratégie permettant d’établir une infection avec succès. Des plantes sur-exprimant RTL2 ou des mutants rtl2 ne montrent aucun défaut morphologique, et ne montrent pas de changement majeur du répertoire des petits ARNs endogènes. Toutefois, la sur-expression de RTL2 augmente l’accumulation des petits ARNs exogènes dans des essais d’expression transitoire, et cette activité nécessite un domaine RNaseIII fonctionnel. Il est donc possible que RTL2 clive certains substrats pour faciliter l’action des enzymes DCL. / Small RNAs, including miRNA and siRNA, play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode four DICER-LIKE (DCL) RNaseIII enzymes. DCL1 produces miRNAs, while DCL2, DCL3 and DCL4 produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Arabidopsis plants over-expressing RTL1 exhibit morphological defects and lack all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. RTL1 activity requires a functional RNaseIII domain. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. Plants over-expressing RTL2 and rtl2 mutants do not exhibit morphological defects and do not show major changes in the endogenous small RNA repertoire. However, RTL2 over-expression enhances the accumulation of exogenous siRNAs in transient assays, and this activity requires a functional RNaseIII domain. Therefore, it is possible that plant RTL2 processes certain substrates to facilitate the action of DCL enzymes.

Le blé tendre ou Triticum aestivum possède un génome hexaploïde (2n=6X=42 chromosomes) de très grande taille (17 Gb) formé de trois génomes diploïdes homéologues. Afin de répondre aux nouvelles contraintes sociétales et environnementales, de nouvelles variétés de blé doivent être créées. L’amélioration des variétés peut se baser sur la variabilité génétique, mais également sur les modifications épigénétiques mises en évidence ces dernières années. La méthylation de l’ADN est l’une de ces modifications, et se retrouve sous forme de 5-méthylcytosine (5mC). Le gène METHYLTRANSFERASE1 (MET1) est bien connu pour son rôle dans le maintien de la méthylation de l’ADN au niveau des 5mC en contexte CpG chez Arabidopsis. Afin d’identifier les orthologues de MET1 chez le blé, la méthode de capture de séquence génomiques sur puce à ADN a été combinée avec des analyses bioinformatiques réalisées sur les récentes données de séquençage du génome du blé. J’ai identifié neufs copies du gène TaMET-1 sur les chromosomes 2, 5 et 7, et ce pour les trois génomes homéologues. Deux évènements de duplications géniques semblent être à l’origine de ces neufs copies. Suite à la seconde duplication, les copies du chromosome groupe 5 (groupe 5) ont évolué plus rapidement pour devenir des pseudogènes, peu ou pas exprimés. L’analyse de données d’expression par RNA-Seq a révélé que les copies des groupes 2 sont 10 à 40 fois plus exprimées que celles du groupe 7. Nous avons montré, pour les régions promotrices des groupes 5 et 7, la relation existant entre un faible niveau d’expression, une forte vitesse évolutive et un enrichissement en CpG, ce dernier étant associé avec une forte méthylation. Plusieurs stratégies ont été envisagées afin de valider les fonctions des gènes TaMET-1 : le crible de population de TILLING (Targeting Induced Local Lesions in Genomes), la construction de lignées RNAi ciblant MET1 et l’utilisation de lignées délétées pour la partie du chromosome portant la copie TaMET-1 la plus exprimée. Aucun mutant de TILLING n’a été identifié. Une analyse par conversion au bisulfite est actuellement en cours sur les lignées RNAi et lignées de délétion afin de valider un effet possible sur la méthylation. Ce travail permettra de conclure sur l’identification de lignées capables de modifier les profils de méthylation et qui pourraient être le point de départ pour induire de la variabilité épigénétique. / Bread wheat or Triticum aestivum possesses a large hexaploid genome (2n=6X=42 chromosomes, 17 Gb) formed by three homeologous diploid genomes. To better respond to new societal and environmental constraints, new wheat varieties have to be created. Breeding can use genetic variability and epigenetics modifications highlighted in recent years. DNA methylation is one of the epigenetic marks and is found as 5-methylcytosine (5mC). The METHYLTRANSFERASE1 gene (MET1) is known to be involve in maintenance of 5mC DNA methylation in CpG context. To identify MET1 genes in wheat, a method based on genomic sequence capture and bioinformatics analyses on data from wheat genome were combined. I identified nine copies of TaMET-1 gene on chromosomes 2, 5 and 7, for the three homeologous genomes. These nine copies seem to originate from two duplication events. After the second one, copies from chromosome 5 (group 5) evolved faster to become pseudogenes, not expressed or at a low level. Analysis of RNASeq expression data revealed that group 2 copies are expressed from 10 to 40 times more than the ones from group 7. For the promoter regions of group 5 and 7, we have shown a relationship between low expression level, high evolution rate and CpG enrichment, which is associated with high DNA methylation level. Several strategies were chosen to validate MET1 gene function : screen of TILLING population, construction of RNAi lines and use of deleted-chromosome line for the most expressed TaMET-1 gene. No TILLING mutants were found. An analysis based on bisulfite conversion is in progress on RNAi lines and deleted lines to validate a putative effect on DNA methylation. This work will permit to identify lines able to modify DNA methylation pattern which will be the starting point to induce epigenetics variability.

O aumento da incidência da doença huanglonbing (HLB) nos pomares brasileiros tem contribuído significativamente para o aumento do custo de produção de laranja no Brasil, devido à suscetibilidade de todas as variedades cultivadas. O HLB é associado às bactérias Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus e Candidatus Liberibacter americanus, transmitidas pelos psilídeos Diaphorina citri e Trioza erytreae. No Brasil, o inseto vetor responsável pela rápida disseminação da doença é o psilídeo Diaphorina citri. Os principais métodos de controle do HLB são a eliminação de plantas sintomáticas, uso de mudas sadias e controle do vetor. O uso de plantas geneticamente modificadas pode ser uma alternativa ao uso excessivo de inseticidas para controle do inseto vetor. Sendo assim, pode-se adotar uma nova tecnologia que se baseia no uso de RNAs de interferência (RNAi) para o silenciamento de genes específicos do inseto vetor. Dessa forma, o objetivo deste trabalho foi avaliar o efeito do silenciamento dos genes calreticulina (DcCRT), lacase (DcLAC) e Snf7 (DcSNF7) de Diaphorina citri por RNA de interferência via alimentação em folhas destacadas de Murraya paniculata que absorveram solução contendo dsRNA de silenciamento. A princípio, foi avaliada a expressão dos genes candidatos em diferentes estádios fenológicos dos insetos. Em seguida, foram realizados bioensaios de sobrevivência e silenciamento, com as doses 0, 5, 25 e 50&#181;g de dsRNA. A sobrevivência dos insetos foi avaliada ao longo de 144 horas, enquanto que os insetos utilizados na análise de expressão gênica foram coletados 12, 24, 48, 72 e 96h após o início da alimentação. Para os genes DcCRT, relacionado à quelatização de íons cálcio, e DcLAC, relacionado à gelificação oxidativa do estilete, ambos envolvidos no processo alimentar do inseto, também foi observado o efeito das doses de dsRNA na quantidade de alimento ingerido, 120h após o início da alimentação. Além disso, também foi realizado bioensaio com dsRNA Cy3-labbled, para verificar a distribuição do dsRNA absorvido. A expressão dos genes candidatos, de modo geral, se manteve uniforme ao longo dos estádios de desenvolvimento dos insetos, exceto para o gene DcLAC que apresentou maior expressão em adultos recém emergidos do que nos demais estádios avaliados. Nos bioensaios com dsRNA de silenciamento, observou-se fenótipo letal apenas com o silenciamento do gene DcSnf7, 144 horas após o início da alimentação. Durante o período avaliado, foi constatada redução na expressão gênica apenas de DcCRT, após 12h de alimentação e DcLAC após 96h de alimentação com seus respectivos dsRNA de silenciamento. Além disso, a dose de 5&#181;g de dsRNA foi suficiente para causar tal efeito de silenciamento gênico. No bioensaio para quantificação da alimentação, observou- se redução na excreção de aminoácidos nos insetos alimentados com 5&#181;g de dsRNA, em ambos os genes candidatos (DcCRT e DcLAC) 120h após o início da alimentação. Adicionalmente, a análise de distribuição de dsRNA pela folha mostrou que há absorção do dsRNA pela folha destacada, permitindo a alimentação pelo inseto. Dessa forma, conclui-se que os genes estudados apresentam potencial para o controle do psilídeo Diaphorina citri, via silenciamento gênico por RNAi. / The increase in the incidence of huanglonbing disease (HLB) in Brazilian orchards has contributed to increase of orange production costs in Brazil, due to the susceptibility of all cultivated variety of orange. HLB is associated with the Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus bacteria, transmitted by the psyllids Diaphorina citri and Trioza erytreae. In Brazil, the insect vector responsible for the rapid spread of the disease is Diaphorina citri. The most important methods of HLB control are the elimination of symptomatic plants, the use of healthy nursery trees, and vector control. The use of genetically modified plants may be an alternative to the excessive use of insecticides to control the insect vector. Therefore, a new technology based on using RNAs of interference (RNAi), for silencing specific genes of the insect vector can be used. Thus, the aim of this work was to evaluate the effect of silencing calreticulin (DcCRT), laccase (DcLAC) and Snf7 (DcSNF7) genes in Diaphorina citri by interference RNA through feeding in leaves of Murraya paniculata that absorbed a solution containing dsRNA of silencing. At first, the expression of the candidate genes in different phenological stages of the insects was evaluated. Survival and silencing bioassays were then performed at doses of 0, 5, 25 and 50 &#181;g of dsRNA. Insect survival was evaluated over 144 hours, while the insects used in gene expression analysis were collected 12, 24, 48, 72 and 96 hours after feeding. For the DcCRT genes, associated to the calcium ion chelation, and DcLAC, related to the oxidative gelling of the stylet, both involved in the insect feed process, the effect of the doses of dsRNA on the amount of food ingested, 120h after the beginning of feeding was observed. In addition, a bioassay with Cy3-labbled dsRNA was also performed to verify the distribution of absorbed dsRNA. The expression of the candidate genes generally remained uniform throughout the stages of insect development, except for the DcLAC gene that showed greater expression in newly emerged adults than in the other evaluated stages. In the bioassays with dsRNA for gene silencing, a lethal phenotype was observed only with the silencing of the DcSnf7 gene, 144 hours after the beginning of feeding. During the evaluated period, there was a reduction in the gene expression only of DcCRT, after 12h of feed and DcLAC after 96h of feeding with their respective silencing dsRNA. Additionally, the dose of 5&#181;g of dsRNA was enough to cause this gene silencing effect. In the bioassay for feeding quantification, a reduction in amino acid excretion was observed in insects fed with 5 &#181;g of dsRNA, in both candidate genes (DcCRT and DcLAC) 120 h after the beginning of feeding. In addition, the analysis of dsRNA distribution by the leaf showed that there is absorption of the dsRNA by the detached leaf, allowing feeding by the insect. Thus, we conclude that the genes studied have potential for the control of the psyllid Diaphorina citri, through gene silencing mediated by RNAi.

A inativação do cromossomo X (ICX) é o fenômeno através do qual um dos cromossomos X das fêmeas de mamíferos é silenciado para atingir compensação de dose em relação aos machos. Ela envolve a expressão do gene XIST exclusivamente no X inativo, e a associação em cis de seu RNA nesse cromossomo. Isso inicia a imposição de várias marcas epigenéticas no cromossomo X inativo, que garantem a manutenção deste estado de silenciamento transcricional de maneira estável durante todas as mitoses num organismo. Uma dessas modificações epigenéticas é a metilação do DNA, desempenhada principalmente pela enzima DNMT1. Os papéis de XIST e DNMT1 na manutenção da inativação do cromossomo X ainda são controversos em humanos, e nesse sentido foi objetivo desse trabalho analisar a possível função desses genes nesse processo em células humanas não transformadas. Foi otimizado um sistema experimental para o estudo de possíveis perturbações na manutenção da inativação do cromossomo X, onde a re-expressão de genes submetidos a esse processo pode ser monitorada. Nesse sistema foram identificados dois genes, MAOA e GYG2, cujo padrão de expressão no X inativo difere do previamente descrito. Demonstrou-se que baixos níveis de expressão do gene XIST foram suficientes para manter seu RNA associado ao X inativo, conservando o estado silenciado desse cromossomo. Além disso, foram obtidos indicativos de que a inibição de XIST em fibroblastos humanos gera uma diminuição da viabilidade celular. Foi possível demonstrar que DNMT1 é necessária para a manutenção da metilação global do genoma em células humanas não transformadas, e que eXISTe um mecanismo de compensação da inibição desse gene que leva ao aumento da expressão de DNMT3B. Ainda se observou que a repressão de DNMT1 não é suficiente para levar à reativação de genes no cromossomo X inativo. Além disso, a desmetilação encontrada nos promotores de MAOA e XIST não foi suficiente para levar à expressão destes genes nos cromossomo X inativo e ativo, respectivamente. Estes resultados enfatizam a necessidade de se estudar os mecanismos moleculares da ICX em humanos utilizando sistemas experimentais adequados para a análise de herança epigenética. / X chromosome inactivation (XCI) is the phenomenon through which one of the X chromosomes in female mammals is silenced to achieve dosage compensation related to males. It involves the expression of XIST gene exclusively from the inactive X, and the association of its RNA in cis in this chromosome. This leads to a series of epigenetic modifications in the chromatin of the inactive X (Xi) that guarantee a stable maintenance of the transcriptional silence through all the mitoses in the organism. One of these epigenetic modifications is DNA methylation, achieved mainly by the maintenance DNA methylase DNMT1. The roles of XIST and DNMT1 in the maintenance phase of XCI are controversial in humans. Therefore, the main goal of this present work was to analyze some of the possible functions of these genes in this process in untransformed human cells. An experimental system was optimized to study possible disturbances in maintenance of XCI, where the re-expression of genes submitted to this process could be monitored. In this system we identified two genes, MAOA and GYG2, whose pattern of expression on the Xi, differed from what had been previously described. It was demonstrated that low levels of XIST expression were sufficient to keep its RNA associated to the Xi, assuring the silenced state of this chromosome. Besides, evidences have been found that XIST inhibition in human fibroblasts reduces cellular viability. It was possible to demonstrate that DNMT1 is necessary to the maintenance of global genome methylation in untransformed human cells, and the eXISTence of a compensation mechanism involving DNMT3B upregulation. It was also observed that repression of DNMT1 was not sufficient to reactivate genes of the Xi chromosome. Additionally, demethylation of MAOA and XIST promoters was not enough to cause expression of these genes on the inactive and active Xs, respectively. All these results emphasize the requirement of studying the molecular mechanisms of XCI in humans using experimental systems appropriate for the analysis of epigenetic inheritance.