Abstract
The modulation of chromatin dynamics by ATP-dependent chromatin remodeling factors has been recognized as an important mechanism to regulate the balancing of self-renewal and pluripotency in embryonic stem cells (ESCs). Here we have studied the effects of a partial deletion of the gene encoding the chromatin remodeling factor Chd1 that generates an N-terminally truncated version of Chd1 in mouse ESCs in vitro as well as in vivo. We found that a previously uncharacterized serine-rich region (SRR) at the N-terminus is not required for chromatin assembly activity of Chd1 but that it is subject to phosphorylation. Expression of Chd1 lacking this region in ESCs resulted in aberrant differentiation properties of these cells. The self-renewal capacity and ESC chromatin structure, however, were not affected. Notably, we found that newly established ESCs derived from Chd1Δ2/Δ2 mutant mice exhibited similar differentiation defects as in vitro generated mutant ESCs, even though the N-terminal truncation of Chd1 was fully compatible with embryogenesis and post-natal life in the mouse. These results underscore the importance of Chd1 for the regulation of pluripotency in ESCs and provide evidence for a hitherto unrecognized critical role of the phosphorylated N-terminal SRR for full functionality of Chd1.
Highlights
Correspondence and requests for materials should be addressed to Embryonic stem cell differentiation requires full length Chd[1]
We found that a previously uncharacterized serine-rich region (SRR) at the N-terminus is not required for chromatin assembly activity of Chd[1] but that it is subject to phosphorylation
Likewise, immunoblotting of bulk H3K9me[3] and H3K4me[3] revealed comparable levels of modification in both cell lines (Figure 3e). It appears that loss of the N-terminal domain does not incapacitate Chd[1] to an extent that results in the increased heterochromatin formation shown before in Chd[1] knock-down ESCs26. Together these results suggest that the SRR at the N-terminus of Chd[1] is not required for embryonic stem cells (ESCs) survival and self-renewal, that it does not affect overall chromatin appearance and that pluripotency gene expression is largely undisturbed
Summary
Correspondence and requests for materials should be addressed to Embryonic stem cell differentiation requires full length Chd[1]. The modulation of chromatin dynamics by ATP-dependent chromatin remodeling factors has been recognized as an important mechanism to regulate the balancing of self-renewal and pluripotency in embryonic stem cells (ESCs). We found that newly established ESCs derived from Chd1D2/D2 mutant mice exhibited similar differentiation defects as in vitro generated mutant ESCs, even though the N-terminal truncation of Chd[1] was fully compatible with embryogenesis and post-natal life in the mouse These results underscore the importance of Chd[1] for the regulation of pluripotency in ESCs and provide evidence for a hitherto unrecognized critical role of the phosphorylated N-terminal SRR for full functionality of Chd[1]. RNAi-driven Chd[1] knock-down in ESCs caused the appearance of more compact heterochromatin foci than in control ESCs, and it was proposed that a global increase in heterochromatin may be responsible for the differentiation defects in the knock-down cells[26]
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