Epitope_tagging_of_Endogenous_Proteins_to_Study_the_Transcriptional_Network_for_Pluripotency_of_Embryonic_Stem_Cells. Epitope_tagging_of_Endogenous_Proteins_to_Study_the_Transcriptional_Network_for_Pluripotency_of_Embryonic_Stem_Cells

The project mainly focuses on the following respects: (1) revealing new components and interactions within a transcriptional network underlying establishment and maintenance of the pluripotent state in mouse embryonic stem (ES) cells, (2) revealing the detailed changes in the transcriptional network as differentiating the mouse ES cells into neural stem (NS) cells. The goal of this project is trying to map the genetic regulatory nodes and networks that control the activity of embryonic stem cells and the process of differentiation to specific cell fate (i.e. neural differentiation). By applying the protein affinity purifications followed by Mass-Spec as well as Chromatin Immunoprecipitation followed by Sequencing (ChIP-Seq), we shall be able to identify the components of our interested protein complexes and their DNA binding patterns in both of ES cells and NS cells. So far this project has established a high throughput methodology for in vivo protein tagging to generate mouse ES cell lines with specific knocked-in affinity tag, which allows the following purification to be highly specific and robust. The processes of evaluating the tag in protein affinity purification and also in chromatin immunoprecipitation experiments have been completed. We conclude that the tag, designing for fulfilling the dual purpose on studying the protein-protein and protein-DNA interactions, performs very well in both protein purification and ChIP experiments. The on-site Illumina/Solexa sequencing technology was applied to identify the DNA sequences obtained from ChIP experiments, with higher resolution, fewer artifacts, greater coverage and a larger dynamic range. The results from ChIP-Seq from ES cells are reproducible and highly agree with published data set. At this moment, we are in the process of validating the DNA binding profiles that we are interested in neural stem cells and making the comparison of the binding patterns between ES cells and NS cells. In order to properly interpret the biological significances of the DNA binding profiles obtained by previous ChIP-Seq experiments, the further identifications on the transition of RNA PolII binding, as well as the transition of epigenetic marks, will be performed, accompanied by the expression profiling by microarray or RNA-Seq technologies. Hence the future works will include: (1) further analyzing the interested protein-DNA binding peaks identified in ES and NS cells, (2) obtaining the data from protein affinity purifications in ES and NS cells, (3) identifying the transition of RNA PolII binding in neural differentiation, (4) identifying the transition of epigenetic marks in neural differentiation, (5) expression profiling by microarray or RNA-Seq, (6) manuscripts preparation.