Abstract
The unique properties of embryonic stem cells (ESC) rely on long-lasting self-renewal and their ability to switch in all adult cell type programs. Recent advances have shown that regulations at the chromatin level sustain both ESC properties along with transcription factors. We have focused our interest on the epigenetic modulator HP1γ (Heterochromatin Protein 1, isoform γ) that binds histones H3 methylated at lysine 9 (meH3K9) and is highly plastic in its distribution and association with the transcriptional regulation of specific genes during cell fate transitions. These characteristics of HP1γ make it a good candidate to sustain the ESC flexibility required for rapid program changes during differentiation. Using RNA interference, we describe the functional role of HP1γ in mouse ESC. The analysis of HP1γ deprived cells in proliferative and in various differentiating conditions was performed combining functional assays with molecular approaches (RT-qPCR, microarray). We show that HP1γ deprivation slows down the cell cycle of ESC and decreases their resistance to differentiating conditions, rendering the cells poised to differentiate. In addition, HP1γ depletion hampers the differentiation to the endoderm as compared with the differentiation to the neurectoderm or the mesoderm. Altogether, our results reveal the role of HP1γ in ESC self-renewal and in the balance between the pluripotent and the differentiation programs.
Highlights
Embryonic stem cells (ESC) are the pluripotent cells that give rise to the differentiated cells of the three germ layers at the earliest stages of development [1]
The role of HP1c in ESC was examined by RNA interference via lentiviral vectors encoding a short hairpin RNA
This observation was confirmed by a proliferation assay that measured BrdU (BromodeoxyUridine) incorporation in newly synthesized DNA during a unique cell cycle and which showed around 20% decrease in DNA synthesis in the shHP1c cell lines compared to the shCTR line (Figure 1D)
Summary
Embryonic stem cells (ESC) are the pluripotent cells that give rise to the differentiated cells of the three germ layers at the earliest stages of development (endoderm, mesoderm and ectoderm) [1]. In mice, these cells are derived from the inner cell mass of blastocysts and are capable of prolonged self-renewal in vitro. ESC maintenance is supported by a conserved and restricted set of key transcription factors, namely Oct4 [2,3], Nanog [4,5] and Sox2 [6,7] Characterization of these cells has shown that the progression from a pluripotent to differentiated status is correlated with chromatin condensation [8] and enrichment in silenced chromatin marks (see [9] for review) through heterochromatin formation. RNA interference screens targeting chromatin-associated proteins have revealed the existence of ESC regulations at the chromatin level that contribute to their open chromatin state and that are important for ESC properties, namely their self-renewal and their pluripotency [12,13]
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