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Functional analysis of genes candidates to maintain the X chromosome inactivation in humans

Grant number: 14/26348-6
Support type:Scholarships in Brazil - Master
Effective date (Start): April 01, 2015
Effective date (End): February 28, 2017
Field of knowledge:Biological Sciences - Genetics
Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal Investigator:Lygia da Veiga Pereira
Grantee:Karla Alejandra Vizcarra Zevallos
Home Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The X Chromosome Inactivation (XCI) in females is an example of epigenetic regulation. The silencing of X chromosomes leads to stable formation of facultative heterochromatin by acquiring multiple changes in chromatin that are maintained in the subsequent cell divisions. The mechanisms involved in the initiation and establishment of XCI have been extensively studied, especially in mice. Nowadays, some epigenetic characteristics associated with maintenance of XCI have been described; however, the mechanisms of action and the identity of the different factors involved in the process are unknown or poorly understood. Our laboratory performed a screening of genomic functional shRNAs libraries to find genes involved in maintaining XCI in humans. From this study, 20 new candidate genes were identified as possibly involved in the maintenance of XCI. The aim of this project is to validate four of these candidates (SRSF10, H3F3B, ASF1A and PSKH1), analyzing the degree of their involvement in epigenetic control process of chromosome X. For this, the silencing of different candidate genes will be accomplished through specific shRNAs in female primary fibroblasts with full deflection of XCI. Then, the reactivation of Xi will be assessed in these cells by the analysis of allele-specific expression of X- linked genes containing informative SNPs previously identified in our laboratory. There will also be performed RNA-FISH assays and trimetilation of histones to evaluate the reactivation of Xi. Finally, if more than one candidate gene presents a role in maintaining the XCI, they will also be evaluated by their action in synergy in the cells. The study proposed here will contribute to a better understanding of the XCI maintenance process and the factors involved in epigenetic inheritance in humans. (AU)