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Análisis de la expresión génica diferencial de células eritroleucémicas resistentes a la diferenciación: relevancia de las proteínas del citoesqueleto de actina

AuthorsFernández-Calleja, Vanessa
AdvisorKrimer, Dora B.
KeywordsCélulas eritroleucémicas
Diferenciación celular
Citoesqueleto de actina
Issue Date2017
PublisherCSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)
Universidad Complutense de Madrid
AbstractFriend murine erythroleukemia cells (MEL) derive from proerythroblasts transformed with the Spleen Focus Forming Virus (SFFV) (Friend et al. 1966; Ruscetti (1999) where integration occurred several kilobases upstream the PU.1 locus (Fernandez-Nestosa et al. 2013). These cells remain in a proliferative state and do not differentiate in the presence of erythropoietin. MEL-DS19 cell line may overcome the blockage, however, and reinitiate differentiation when exposed to a number of different chemical agents, such as hexamethylene-bis-acetamide (HMBA). This feature makes MEL-DS19 cells an extremely useful model to study reprogramming of tumor cells to a non-malignant phenotype and to analyze the mode of action of different chemotheraupetics compounds. We previously reported the establishment of HMBA-resistant cell lines (MEL-R) that are unable to differentiate, even in the presence of the inducer (Fernandez-Nestosa et al. 2008). On the other hand, the network of actin filaments provides mechanical support to the cell cytoskeleton, but it is increasingly acknowledged that it also contributes to other critical cellular processes. Emerging evidence points to a role for the actin cytoskeleton in controlling and regulating receptor signaling (Mattila et al. (2016). In this line, changes in the regulation of actin cytoskeleton could be implicated in the blockade of the erythroid differentiation program. The aims of this thesis were: -Identify genes whose differential expression is higher in MEL-DS19 in relation to MEL-R, and viceversa by using RNA-seq.
-Analyze specific hematopoietic genes whose functions are related to the organization and polymerization of the actin cytoskeleton, i.e. Was (Wiskott-Aldrich syndrome), Btk (Bruton’s tyrosine kinase) and Plek (pleckstrin). - Generate genomic deletions of Was, Btk and Plek in MEL-DS19 cells via CRISPR/Cas9. - Established MEL-R transfectants that overexpressed those same genes. -Analyze the possible implication of increased expression of histone-encoding genes in the resistant line MEL-R. Our results can be summarize as follows: Resistant cells (MEL-R) showed phenotypic differences in relation to the parental cell line. We corroborated a significant increase in the size of MEL-R cells that parallel a prolonging doubling time. The distribution of MEL-R cells along the cell cycle follows the same pattern observed in uninduced MEL-DS19 cells. However, we found that the DNA content in the resistant lines has doubled, generating tetraploid cells. This data supports the idea that tetraploid cells enhance tumorigenic capacity relative to diploid cells. In an attempt to identify genes related to the resistant phenotype we used high-throughput RNA sequencing (RNA-seq) to compare the transcriptomes of the erythroleukemia progenitor and the resistance cell lines. RNA-seq revealed a total of 596 genes with a p-value adjusted less than 0.05 that were differentially expressed by more than two-fold, of which 486 genes were up-regulated in MELDS19 cells and 110 in MEL-R cells, enlightening that the number of genes expressed in the parental cell line decreased as the cells acquired the resistant phenotype. The progressive gene silencing observed in MEL-R cells involves several mechanisms. We proved that heterochromatinization, a marker for transcription down-regulation, is enhanced in MEL-R cells relative to undifferentiated MEL-DS19 cells, but is nevertheless lower than in HMBA-differentiated cells. We also examined the methylation status of Was, Btk and Plek genes and found that in MEL-R cells the CpG islands remained methylated in contrast to their non-methylated status in MEL-DS19 and HMBA-differentiated cells.
The expression pattern of enzymes that catalyze DNA methylation and demethylation revealed an up-regulation of Dnmt1 and Tet3 expression in MEL-R cells. These results showed that the increase in DNA methylation by Dnmt1 in MEL-R cells overlaps with a decrease in demethylation by Tet3, which presumably results in the silencing of gene promoters. RNA-seq analysis showed highest differential expression allowed identification of a group among genes up-regulated in MEL cells. These genes are related with the organization of the actin cytoskeleton network, the majority of these genes are preferentially expressed in the erythroid lineage. Among this group stand out Bruton's tyrosine kinase (Btk), Wiskott Aldrich syndrome (Was) and pleckstrin (Plek). In order to study the potential implications of these genes on resistance to cell differentiation we performed genome deletions of Was, Btk or Plek in MEL-DS19 based on CRISPR/Cas9 system. In the first two cases, deficiencies were observed in the organization and polymerization of the actin cytoskeleton as opposed to the results obtained when Plek was deleted. The ability of the resistant line to reverse the wild phenotype was also determined. We established stable transfectants expressing each of the proteins in MEL-R cells. We observed a recovery in the organization of the actin cytoskeleton in Was and Btk. Moreover, we demonstrated that Was and Plek regulate the transcription expression of Btk, function that could be independent of the organization of the actin cytoskeleton. Altogether, these results suggest that disturbances of genes involved in the regulation and polymerization of actin cytoskeleton are coupled to a resistant differentiation phenotype.
Description131 p.-49 fig.-6 tab.-anexo
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