Browsing by Author "Silva, Leonardo Mendes e"
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- From pluripotency to early events leading to cardiogenesisPublication . Silva, Leonardo Mendes e; Bragança, José; Martinho, Rui GonçaloHeart diseases are the leading cause of death worldwide, which includes acquired cardiovascular diseases and congenital heart diseases. The Cited2 gene is important for proper heart development and cardiac cell differentiation from pluripotent stem cells. Previous studies suggested that the role of Cited2 in cardiac differentiation could begin during the early events that occur in pluripotent cells that undergo differentiation. In mouse embryonic stem cells, Cited2 is important for self-renewal maintenance, as cells die or differentiate under Cited2-depleted conditions. In differentiation, embryonic stem cells depleted of Cited2 at the onset of differentiation show an impairment of cardiac cell differentiation. However, the mechanisms behind these observed phenotypes of Cited2-depletion are not well established. In this work, differentially expressed genes were identified in undifferentiated mouse embryonic stem cells and differentiated cells on day-4 of differentiation. Thus, using mouse embryonic stem cells that can be conditionally depleted of Cited2, after 16 hours of incubation it was observed that Cited2-depleted cells under undifferentiation conditions tend to show increased levels of the DNA damage marker γH2AX, concomitant with decreased expression of DNA repair genes (Rad51c, Rad9b, and Mdc1) and increased expression of pro-apoptotic genes (p21, Ptges, Plk2). In differentiation conditions using the hanging drop method, on day-4 of differentiation, epigenetic mark H3K27ac showed a decrease in the promoter region of cardiopoietic genes concordant with their downregulation (Brachyury, Cdx2, Dkk1, Isl1, Kdr, Mesp1, Wnt5a). However, the results of the H3K27me3 marks, showed higher variability and did not match the reciprocal marks of H3K27ac. Moreover, the increase in H3K27ac mark in undifferentiated Cited2-depleted cells corresponded, as expected, to an upregulation of the same genes. Lastly, histone acetyltransferase activity assay using the whole cell extract showed a tendency to increase acetylation in Cited2-depleted cells. In conclusion, the role of Cited2 in DNA repair and the cause of increased cell death remains to be established, while delayed expression of mesoderm/cardiac genes could be associated with misregulation of epigenetic H3K27(me3/ac) marks at the promoters of cardiopoietic genes.