Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment.

Embryonic stem cells (ESCs) hold a great potential for cell replacement, despite a number of significant hurdles that still stand in the way of the realization of that potential. The most important among these are differentiation efficiency, safety concerns and overcoming immune rejection.1 Currently, ESCs and their resulting differentiated cell types provide a promising in vitro system for the study of early human embryonic development.2, 3 As such, they can be used as cell lines with genetic disorders4, 5 and are being used increasingly in drug screening.6, 7 Differentiation of ESCs requires the repression of transcription factors involved in maintaining pluripotency and the activation of developmental genes. Both processes are directed by specific epigenetic mechanisms. An example of the first process is the promoter-methylation-dependent repression of Nanog and Oct3/4 as cells differentiate.8 Thus far, the activation of developmental genes during DNA-demethylation-induced stem cell differentiation has been less thoroughly studied. Instead, these developmental genes have been reported as being in a repressed state during the early stages of development because of the establishment of specific patterns of histone modifications, which consist of large regions of H3-Lys27 methylation harbouring smaller regions of H3-Lys4 methylation.9 This repressive chromatin state is mediated by the Polycomb group of proteins.10, 11, 12 Besides nitric oxide (NO)13 many epigenetic compounds efficiently invert genes' methylation status and histone patterns; such compounds are currently in use for the treatment of cancer.14 Because treatment of ESCs with 5′-aza-2′-deoxycytidine (AzadC) initiates cardiac differentiation and gene reactivation,15, 16 we tested the potential effect of zebularine (1-(β-𝒟-ribofuranosyl)-1,2-dihydropyrimidin-2-1) on ESCs. Zebularine is a cytidine analogue that is able to inhibit DNA methyltransferases (DNMTs) through covalent binding17, 18, 19 and is a robust inhibitor of cytidine deaminase, a central enzyme for the metabolism of cytosine nucleoside analogues such as AzadC,20 which is increased in tumour cells as a resistance mechanism to antineoplastic drugs. Zebularine acts during DNA replication, substituting cytosine residues following its phosphorylation and conversion to deoxynucleotide. Once incorporated into DNA, it forms covalent complexes with DNMTs leading to inhibition of its activity, thereby decreasing DNA methylation.19 Overall, zebularine is a genome-wide demethylating agent with increased activity in proliferative cells. Unlike AzadC, which is toxic in vitro and in vivo and is unstable in aqueous solution, zebularine is chemically stable in neutral and basic media.21, 22 In addition, zebularine has a smaller myelosuppressive effect than AzadC; this leads to minimal side effects, making zebularine a candidate drug for long-term tumour treatment by oral administration.22 We tested the effect of zebularine on mouse ESCs (mESCs) and detected rhythmic and synchronized beating areas in embryoid bodies (EBs). We next studied the gene and protein expression of cardiac markers, finding that zebularine-treated cells highly expressed cardiac-restricted markers and expressed low levels of pluripotency factors. Moreover, when gene expression was compared between cells treated with zebularine, AzadC and NO, the cardiac expression patterns showed that zebularine preferentially drives the differentiation of mESCs towards a cardiomyocyte-like phenotype. These findings support the hypothesis that zebularine regulates mesodermal differentiation more efficiently than the other drugs tested. Next, we analyzed methylation gene promoter status and detected that Nkx2.5, an early marker of the cardiac lineage differentiation program, was clearly unmethylated and thus transcriptionally activated following zebularine treatment. To decipher the global effect of zebularine on gene expression, we performed microarray analysis and found a significant number of differentially expressed genes with a B-statistic >1, demonstrating that the cellular transcriptome is clearly modified following zebularine treatment. The differentially expressed genes were selected using a linear model approach23 and implemented in the Linear Models for Microarray Bioconductor package and Ingenuity's Pathway Analysis (IPA) software. In addition, experiments having significant signatures or reverse/antisignatures were dissected using the entire mouse gene expression omnibus (GEO) experiments on the mouse Affymetrix platform deposited in the NCBI's GEO database. Moreover, proteins showed different patterns when subjected to two-dimensional differential-in-gel-electrophoresis (2D-DIGE). Finally, we studied the effect of zebularine on human ESCs (hESCs) and observed differences in the expression levels of some cardiac-specific genes after treatment.