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| Author(s): |
Karla Alejandra Vizcarra Zevallos
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2017-07-12 |
| Examining board members: |
Lygia da Veiga Pereira Carramaschi;
Uirá Souto Melo;
Angela Maria Vianna Morgante;
Patrícia Severino
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| Advisor: | Lygia da Veiga Pereira Carramaschi |
| Abstract | |
The X chromosome Inactivation (XCI) in females is an example of epigenetic regulation. Silencing of one of the X chromosomes leads to the stable formation of the facultative heterochromatin through the acquisition of multiple modifications in the chromatin that are maintained in the subsequent cell divisions. Currently, some epigenetic features associated with the maintenance of XCI have been described. Nonetheless, the mechanisms of action and the identity of the different factors involved in the maintenance of XCI are still unknown or poorly understood. Our laboratory performed a genomic functional screening by shRNA (short harpin RNAs) libraries to find genes involved in the maintenance of XCI in humans. From this study, we identified 20 new candidate genes to be involved in the maintenance of XCI. Thus, the objective of this work was to validate the degree of involvement of two of these candidate genes (H3F3B and ASF1A) in the epigenetic process control of the X chromosome. For this, the silencing of the candidate genes was performed in female heterozygous primary fibroblasts for a mutation of the HPRT gene and with a total XCI shift through the use of lentiviral particles carrying specific shRNAs, where the only normal allele of the HPRT gene is in the Xi (inactivated X). Xi reactivation was evaluated in these cells by culturing them in HAT medium, which selects HPRT + cells. Only the fibroblasts that were silenced for the H3F3B gene survived. Furthermore, the cells transduced with shH3F3B.2 express the HPRT wild gene allele, present in Xi, in addition to the mutant gene. RNA-FISH and histone trimethylation assays were performed on these cells to evaluate the loss of inactive chromatin marks. A loss of the XIST cloud was observed in cells transduced with shH3F3B.2 and selected in HAT at high passages. Finally, allele-specific expression analyzes of X-linked genes showed that two genes that undergo XCI showed expression of the inactivated allele (FLNA and FHL1). However, a change in allele-specific expression pattern was also observed in autosomal genes. Finally, the X chromosome general expression analyses showed that cells transduced with shH3F3B.2 and selected on HAT had increased gene expression relative to the control. In conclusion, our results suggest a decondensation of the chromatin in the Xi chromosome and therefore a probable involvement of the H3F3B gene in the maintenance of ICX. (AU) | |
| FAPESP's process: | 14/26348-6 - Functional analysis of genes candidates to maintain the X chromosome inactivation in humans |
| Grantee: | Karla Alejandra Vizcarra Zevallos |
| Support type: | Scholarships in Brazil - Master |