Luciana R. Vasques

Ribozymes and the anti-gene therapy: how a catalytic RNA can be used to inhibit gene function

Ribozymes are RNA molecules that possess the dual properties of RNA sequence-specific recognition and site-specific cleavage of other RNA molecules. These properties provide powerful tools for studies requiring gene inhibition, when the DNA sequence is known. The use of these molecules goes beyond basic research, with a potential impact in therapeutical practice in medicine in the near future. In this review, we briefly describe the progress towards developing this class of molecules and its applications for the control of gene expression.

MicroRNAs: modulators of cell identity, and their applications in tissue engineering

MicroRNAs post-transcriptionally regulate the expression of approximately 60% of the mammalian genes, and have an important role in maintaining the differentiated state of somatic cells through the expression of unique tissue-specific microRNA sets. Likewise, the stemness of pluripotent cells is also sustained by embryonic stem cell-enriched microRNAs, which regulate genes involved in cell cycle, cell signaling and epigenetics, among others. Thus, microRNAs work as modulator molecules that ensure the appropriate expression profile of each cell type. Manipulation of microRNA expression might determine the cell fate. Indeed, microRNA-mediated reprogramming can change the differentiated status of somatic cells towards stemness or, conversely, microRNAs can also transform stem- into differentiated-cells both in vitro and in vivo. In this Review, we outline what is currently known in this field, focusing on the applications of microRNA in tissue engineering.

X-chromosome inactivation: lessons from transgenic mice.

X-chromosome inactivation (XCI) is the process by which mammals perform dosage compensation of X-linked gene products between XY males and XX females, resulting in the transcriptional silencing of all but one X chromosome per diploid cell. XCI involves counting the X chromosomes in a cell, randomly choosing those to be inactivated, spreading the inactivation signal in cis throughout the chromosome, and maintaining the inactive state of those X chromosomes during cell divisions thereafter. How the cell performs all these tasks is a fascinating problem and, together with epigenetic inheritance, a basic cellular mechanism that remains to be fully understood. In this review, we describe recent experiments aimed at understanding the first events of XCI and propose a model for initiation of XCI.

A microduplication of 5p15.33 reveals CLPTM1L as a candidate gene for cleft lip and palate

We report a 10-year-old boy with syndromic cleft lip and palate (CLP) and neuro-psychomotor developmental delay. Oligoarray comparative genomic hybridization (aCGH) detected an approximately 300 kb interstitial microduplication at 5p15.33 encompassing 5 protein-coding genes, including TERT and CLPTM1L, and two microRNA genes. Our findings suggest that the duplicated segment predisposes for cleft lip with or without cleft palate (CL/P), or any of the other phenotypic features presented by the patient. A gene coding a similar protein (CLPMT1) has been implicated in CLP etiology both through linkage studies and by a translocation disrupting the gene, indicating the possible involvement of CLPTM1L with CL/P. This is the first report of a possible connection between CLPTM1L and CLP.

Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.

The Roles of miR-26, miR-29, and miR-203 in the Silencing of the Epigenetic Machinery during Melanocyte Transformation

The epigenetic marks located throughout the genome exhibit great variation between normal and transformed cancer cells. While normal cells contain hypomethylated CpG islands near gene promoters and hypermethylated repetitive DNA, the opposite pattern is observed in cancer cells. Recently, it has been reported that alteration in the microenvironment of melanocyte cells, such as substrate adhesion blockade, results in the selection of anoikis-resistant cells, which have tumorigenic characteristics. Melanoma cells obtained through this model show an altered epigenetic pattern, which represents one of the first events during the melanocytes malignant transformation. Because microRNAs are involved in controlling components of the epigenetic machinery, the aim of this work was to evaluate the potential association between the expression of miR-203, miR-26, and miR-29 family members and the genes Dnmt3a, Dnmt3b, Mecp2, and Ezh2 during cells transformation. Our results show that microRNAs and their validated or predicted targets are inversely expressed, indicating that these molecules are involved in epigenetic reprogramming. We also show that miR-203 downregulates Dnmt3b in mouse melanocyte cells. In addition, treatment with 5-aza-CdR promotes the expression of miR-26 and miR-29 in a nonmetastatic melanoma cell line. Considering the occurrence of CpG islands near the miR-26 and miR-29 promoters, these data suggest that they might be epigenetically regulated in cancer.

Genetic and Epigenetic Effects of Environmental Mutagens and Carcinogens

1Department of Health Sciences, University of Genoa, Via Antonio Pastore 1, 16132 Genoa, Italy 2Toxicology Institute, Preventive Medical College, Third Military Medical University, Chongqing 400038, China 3Department of Genetics and Evolutionary Biology, Institute of Bioscience, University of Sao Paulo, 05508-090 Sao Paulo, SP, Brazil 4Laboratory of Toxicology UT-BIORAD, ENEA, CR Casaccia, Via Anguillarese 301, 00123 Rome, Italy

Knockdown of E2f1 by RNA interference impairs proliferation of rat cells in vitro

E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell proliferation. Thus, blocking E2F1 expression by RNA interference represents a promising therapeutic approach. In this study, the introduction of specific short hairpin RNAs (shRNAs) reduced E2f1 expression by up to 77%, and impaired rat glioma cell proliferation by approximately 70%, as compared to control cells. Furthermore, we investigated the expression of E2f1 target genes, Cyclin A and Cyclin E. Cyclin A was found to be down-regulated, whereas Cyclin E had similar expression to control cells, indicating that gene(s) other than E2f1 control its transcription. Other E2f family members, E2f2 and E2f3, which have been classified in the same subgroup of transcriptional activators, were also analyzed. Expression of both E2f2 and E2f3 was similar to control cells, showing no cross-inactivation or up-regulation to compensate for the absence of E2f1. Nevertheless, their expression was insufficient to maintain the initial proliferation potential. Taken together, our results suggest that shE2f1 is a promising therapy to control tumor cell proliferation.

DNA methylation landscape of hepatoblastomas reveals arrest at early stages of liver differentiation and cancer-related alterations

Hepatoblastomas are uncommon embryonal liver tumors accounting for approximately 80% of childhood hepatic cancer. We hypothesized that epigenetic changes, including DNA methylation, could be relevant to hepatoblastoma onset. The methylomes of eight matched hepatoblastomas and non-tumoral liver tissues were characterized, and data were validated in an independent group (11 hepatoblastomas). In comparison to differentiated livers, hepatoblastomas exhibited a widespread and non-stochastic pattern of global low-level hypomethylation. The analysis revealed 1,359 differentially methylated CpG sites (DMSs) between hepatoblastomas and control livers, which are associated with 765 genes. Hypomethylation was detected in hepatoblastomas for ~58% of the DMSs with enrichment at intergenic sites, and most of the hypermethylated CpGs were located in CpG islands. Functional analyses revealed enrichment in signaling pathways involved in metabolism, negative regulation of cell differentiation, liver development, cancer, and Wnt signaling pathway. Strikingly, an important overlap was observed between the 1,359 DMSs and the CpG sites reported to exhibit methylation changes through liver development (p<0.0001), with similar patterns of methylation in both hepatoblastomas and fetal livers compared to adult livers. Overall, our results suggest an arrest at early stages of liver cell differentiation, in line with the hypothesis that hepatoblastoma ontogeny involves the disruption of liver development. This genome-wide methylation dysfunction, taken together with a relatively small number of driver genetic mutations reported for both adult and pediatric liver cancers, shed light on the relevance of epigenetic mechanisms for hepatic tumorigenesis.Hepatoblastomas are uncommon embryonal liver tumors accounting for approximately 80% of childhood hepatic cancer. We hypothesized that epigenetic changes, including DNA methylation, could be relevant to hepatoblastoma onset. The methylomes of eight matched hepatoblastomas and non-tumoral liver tissues were characterized, and data were validated in an independent group (11 hepatoblastomas). In comparison to differentiated livers, hepatoblastomas exhibited a widespread and non-stochastic pattern of global low-level hypomethylation. The analysis revealed 1,359 differentially methylated CpG sites (DMSs) between hepatoblastomas and control livers, which are associated with 765 genes. Hypomethylation was detected in hepatoblastomas for ~58% of the DMSs with enrichment at intergenic sites, and most of the hypermethylated CpGs were located in CpG islands. Functional analyses revealed enrichment in signaling pathways involved in metabolism, negative regulation of cell differentiation, liver development, cancer, and Wnt signaling pathway. Strikingly, an important overlap was observed between the 1,359 DMSs and the CpG sites reported to exhibit methylation changes through liver development (p<0.0001), with similar patterns of methylation in both hepatoblastomas and fetal livers compared to adult livers. Overall, our results suggest an arrest at early stages of liver cell differentiation, in line with the hypothesis that hepatoblastoma ontogeny involves the disruption of liver development. This genome-wide methylation dysfunction, taken together with a relatively small number of driver genetic mutations reported for both adult and pediatric liver cancers, shed light on the relevance of epigenetic mechanisms for hepatic tumorigenesis.

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.