Epigenética em doenças e desenvolvimento

Dr. Lalande’s research is on the role of epigenetics in disease and development. Epigenetics refers the study of heritable changes in gene function that occur without an alteration in DNA sequence. The Lalande laboratory is focused on translational studies of human epigenetic disorders using murine models and human induced pluripotent stem (iPS) cell technology.

Angelman syndrome is a neurogenetic disorder characterized by severe mental retardation, "puppet-like" ataxic gait with jerky arm movements, seizures, EEG abnormalities, hyperactivity and bouts of inappropriate laughter. Individuals with AS lack a normal maternal copy of the gene encoding ubiquitin protein ligase E3A (UBE3A). UBE3A is transcribed only from the maternal allele in brain with the paternal copy of UBE3A being silenced due to an epigenetic phenomenon called genomic imprinting. Genes that are subject to genomic imprinting are expressed exclusively from one parental allele. This process is said to be epigenetic because it involves heritable changes in gene function that occur without a change in the sequence of DNA.

a) The role of Ube3a in the mammalian stress response. How the loss of UBE3A in brain causes AS is not clear. To study this problem, we have derived two different stable mouse cell lines with shRNA-mediated Ube3a knockdown. Knockdown of Ube3a in both NIH3T3 and P19 embryonic carcinoma cells resulted in increased resistance to both doxorubicin- and paraquat-induced cell death thus indicating that UBE3A functions to promote cell death in response to genotoxic and oxidative stress. Ectopic expression of wild type but not a mutant form of UBE3A, restored doxorubicin sensitivity to Ube3a-deficient NIH3T3 cells, suggesting that the ubiqitin ligase activity of UBE3A is essential for regulating the genotoxic cell death response. We have also generated a mouse model with a "humanized" Ube3a mutation consisting of a 2 bp deletion in the coding region. Using primary cells derived from these mice, we have observed that sensitivity to doxorubicin-mediated genotoxic stress is directly related to the levels of UBE3A protein in murine embryonic fibroblasts (MEFs) derived from mice homozygous, heterozygous and wild type for the Ube3a mutation. Given these results, we conclude that UBE3A may function in the ubiquitin stress response pathway and that the phenotypic manifestations of AS may be due, at least in part, to increased levels of free ubiquitin in the brain.

b) Induced pluripotent stem (iPS) cells models of AS. The recent discovery of genomic reprogramming of human somatic cells into induced pluripotent stem cells (iPS) offers an innovative and relevant approach to the study of human genetic and neurogenetic diseases such as Angelman syndrome. By reprogramming somatic cells from patient samples, cell lines can be isolated that self-renew indefinitely and have the potential to develop into multiple different tissue lineages. Additionally, the rapid progress of research on human embryonic stem cells (hESCs) has led to the development of sophisticated in vitro differentiation protocols that closely mimic mammalian development. We have perfected the experimental approaches for somatic cell reprogramming by introducing four reprogramming factors via retroviral vectors into dermal fibroblasts. We are deriving iPS cells from dermal fibroblasts from normal control and Angelman syndrome patients with the goal of

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