Decoding the contribution of transcription factors to cell fate determination

• It is well accepted that transcription factors (TFs) play a crucial role in determining cell identity. Although RNA expression or protein abundance data show that a large fraction of the total TFs are expressed in a given cell, however, only a small set of them is essential for specifying cell identity. This was elegantly demonstrated through reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) by means of ectopic expression of only four key TFs, Oct-3/4 (Pou5f1), Sox2, Klf4 and c- Myc. In order to decipher the most predominant TFs in specific cell types, we developed a novel massively parallel protein activity assay, Active TF Identification (ATI) that measured DNA-binding activity of TFs in the cell nucleus. This method indicated that around 15 TFs have the highest DNA-binding activities, among which there are “common” TFs universally active in most cell types, “shared” TFs which are active in several cell types and “specific” TFs which are active in only one or two cell types. It has been well established that the gene transcription is highly correlated with disruption of nucleosomes at the gene regulatory elements. In order to test if TFs are the major determinant of chromatin accessibility, we compared the ATI data with the DNase I hypersensitive sites (DHSs) from the same cell or tissue type, and found out that the enriched subsequences in the ATI results are also enriched within the DHSs compared with the non-DHS regions. This suggested that the DNA-binding activity of TFs, especially the most active ones, played major roles in determining the chromatin accessibility. In addition, we also performed the ATI assay using nucleosomal DNA to determine the “pioneer” TFs in cells that are capable of binding condensed chromatin. This study has generated a deeper understanding of the gene regulatory logic and helped us to decipher important TFs in specific cell types.

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