Dissecting the role of linker histone H1 variants in embryonic stem cells
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To further dissect the role of linker histone H1 in embryonic stem cells (ESCs), here, we have utilized a functional reconstitution approach to identify the regions of H1 proteins that are important to mediate neurite outgrowth during neural differentiation of ESCs. We first generated H1 reconstituted ESC lines by overexpressing exogenous H1d proteins in H1 depleted ESCs. We find that overexpression of H1d in H1 depleted ESCs significantly restored the neurite outgrowth capacity of embryoid bodies (EBs) formed from ESCs. Next, to dissect the role of individual domains of H1d in ESC differentiation, we constructed a series of vectors to express H1 deletion mutants in H1 depleted ESCs. Our results show that reconstitution with H1d-GD and H1d-NTD-GD increases neurite outgrowth of EBs, suggesting that the globular domain of H1d is critical in mediating the neurite outgrowth during neural differentiation of ESCs. Lastly, we investigated the potential role of H1 modifications in ESC differentiation. We constructed an expression vector encoding the H1d mutant (H1dK46R) containing a lysine-to-arginine mutation at site K46, and expressed H1dK46R in H1c/H1d/H1e triple knockout (H1 TKO) ESCs by stable transfection and analyzed chromatin binding and biochemical properties through HPLC analysis. Our results suggest that K46R mutation of H1d disrupts the function of H1d in mediating the neurite outgrowth of EBs, suggesting a critical role of post-translational modification(s) on H1d K46 in ESC differentiation. To further dissect the potential mechanisms underlying the defects of H1dK46R mutant, we set out to characterize and compare the mobility of H1dK46R and H1d in ESCs in vivo using fluorescent recovery after photobleaching (FRAP) assay. Results from FRAP assay suggest that K46R mutation decreases dynamic mobility of H1d in ESCs, which may partially contribute to the defects of H1dK46R in mediating proper ESC differentiation. In summary, through a series of studies aimed at dissection of different regions and sites of H1d, we pinpoint GD as a key domain of H1 in mediating neurite outgrowth during neural differentiation of ESCs. Furthermore, our results suggest that the modification(s) on K46 of H1d are critical for proper ESC differentiation and are likely to increase the dynamic plasticity of H1d.