Abstract
Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells.
Highlights
Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve
We inject plasmid DNA, which has binding sites for the factor and an expression reporter, directly into the oocyte germinal vesicle (GV), so that it is immediately delivered to the nuclear environment of the Biochemical experiments have concluded that the dwell time of a transcription factor on its specific DNA site is remarkably short, on the order of seconds [1,2,3,4,5], and may involve oscillation of binding
It would be of considerable interest if the dwell time for some kinds of transcription factors were found to be enormously longer under certain circumstances
Summary
Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl for nerve. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl transcription factor protein. | | | | Xenopus transcription factor oocytes dwell time Ascl gene transcription were and include Ascl, a major neurogenic inducer in embryonic cells and one which causes cells to follow a neural differentiation pathway [7, 8]. It can make adult cells of different kinds become neural [9], like MyoD does for muscle [10]. This may enable this factor to both initiate and stabilize a differentiated state and so give new understanding of the mode of action of a transcription factor in some kinds of cells
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