Molecular regulation of Sox2 expression during differentiation of chick embryonic stem cells

摘要

The transcription factor Sox2 has a key role not only in maintaining stem stateudbut also in specification of neural fate of embryonic cells. Multiple regulatoryudelements have been identified in the Sox2 locus (Uchikawa et al, 2003). In theuddeveloping embryo, these regulatory elements are activated differentially inudtime and space. We studied the activity of 25 defined regulatory elements ofudthe Sox2 promoter in three different lines of chick ES cells. By transfection ofudplasmids encoding Enhanced Green Fluorescent Protein (EGFP) and theudminimal promoter thymidine kinase (tk) coupled with individual Sox2 regulatoryudelements we find that the Sox2 enhancer N2 has the highest activity inudproliferating chick cell lines compared with other enhancer regions. Underudconditions that induce ES cells to differentiate into neurons the activity of theudN2 enhancer increased along with an increase in levels of expression of Sox2udmRNA. Further analysis of the N2 enhancer sequence identified two subregionsudwith 176 and 73 base pairs (bp) which are highly conserved betweenudchick, mouse and man. Functional studies performed with the tk-EGFPudreporter plasmids under the control of five regulatory sequences containing theudmouse N2 enhancer in its full length, its two sub-regions (176 and 73 bp) orudsequences composed of the full length of the mouse N2 from which each ofudthe two sub-regions 176 bp and 73 bp has been deleted confirmed that theudtwo sub-regions of the N2 enhancer account for its activity in both proliferatingudcES cells as well as their induced neural differentiation state. These findingsudsuggest that N2 core regulatory regions encode conserved instructionsudrequired to direct expression of Sox2 both in embryonic stem cells induced toudneural differentiation and in the neural plate of the embryo itself.