Abstract
Human embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation.
Highlights
Human embryonic stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development
Of the 500 genes differentially expressed between D12 and D20, almost 55% were down regulated on D20 when compared to D0 justifying the formation of mature counterparts on D20 compared to D12
Of all the genes being altered during ES cells differentiation, transcription factor NR2F2 reported to repress OCT-4 during neural differentiation[32,33], has not yet been reported during cardiac differentiation but its mutation is known to cause atrial septal defects[31]
Summary
Human embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20 This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. In order to address these questions, it is necessary to understand stage-wise gene expression during early cardiac development and hES cells serve as an excellent model to study this differentiation in vitro. A study by Murry’s group determined the evolution of chromatin modifications like H3K4me[3] and H3K27me[3] along cardiac lineage formation wherein the dynamic acquisition of H3K4me[3] and H3K27me[3] by various transcription factors during differentiation at the five different time points was identified[18]. Bruneau and colleagues[19] revealed a unique epigenetic pattern of H3K27ac, H3K4me[1], H3K4me[3] and H3K4me[3] during the gene expression changes of cardiomyocytes differentiation in vitro
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