Unique DNA binding specificity of Oct-3/4 and its ability to maintain ES in pluripotent state

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ES cells have omnipotent property which can be differentiated to any types of cells and it has been shown that the embryonic Octamer factor, Oct-3/4 plays a central role in maintaining this ES cell specific property. As Oct-1 and Oct-6 proteins are present in ES cells, this fact indicates that Oct-3/4 protein, but not other Octamer factors, has some unique biochemical characteristics which is involved in this biological property of the cells. In this project, I have performed several experiments in order to know what this Oct-3/4 specific biochemical characteristics is. At first, I have mapped the Oct-3/4 domains involved in maintaining pluripotent property of ES cells by using a series of chimeric proteins composed of Oct-3/4 and Oct-6. These analyses revealed that the linker portion and one particular amino acid in POU specific domain are essential, but other portions such as transcriptional activation domains can be replaced by corresponding portions of Oct-6 protein. ES cells beari … More ng the above chimeric protein instead of the wild-type Oct-3/4 express undifferentiated markers such as Rex-1 and UTF1. Furthermore, we confirmed that these cells retains one of the most important properties of ES cells, i.e., the ability to convert to any different types of ectodermal, mesodermal, and endodermal cells.I have also systematically determined DNA binding specificity of Octamer factors as well as the above chimeric proteins by gel shift assay. These analyses led to the identification of 9 different nucleotide sequences which are able to serve as specific or preferential binding sites for Oct-3/4. Furthermore, I have pursued the possibility that this unique DNA binding specificity of the Oct-3/4 is related to its ability to maintain ES cells in pluripotent state. These analyses indeed reveal that there is perfect correlation between the ability to bind to one particular sequence, 5'-ACTAGCAT-3' and that to keep ES cells in undifferentiated state. Interestingly, this sequence is the one which is present in the UTF1 regulatory region and plays central role in supporting UTF1 expression in ES cells.From these results, I concluded that Oct-3/4 downstream genes can be classified into 2 different groups, i.e., one bearing consensus Octamer sequences in their regulatory regions and the other carrying the above variant Octamer sequences in their enhancers and crucial requirement of Oct-3/4 in ES cells is due to its unique ability of this protein to support expression of second group of Oct-3/4 downstream genes. Less