The goal of my graduate work is to address the question of how cell type specific genes become activated during cellular transdifferentiation. In order to increase efficiency of transdifferentiated cells for regenerative therapies; our knowledge of the mechanisms that control cell transdifferentiation and cell type gene activation needs to be greatly expanded. In eukaryotic cells, DNA compaction of chromatin serves as a mechanism for gene regulation by modulating the accessibility of transcription factors (TFs) to DNA. However, the mechanism by which TFs initially engage in the genome and organize it to allow specific cell type decisions remains to be elucidated. Our lab and others have shown that Foxa, a liver-specific factor, binds in early stages of endoderm formation to a nucleosome in the albumin enhancer. Thereby Foxa acts as a pioneer factor, binding nucleosomes, opening local chromatin and allowing binding of subsequent transcription factors. Identification of transdifferentiation factors with pioneer activity similar to Foxa, thus provides a crucial tool inthe understanding of the initial steps in cell transdifferentiation. Based on our lab's previous discoveries and expertise I propose to: (1) identity TFs that act as pioneer initiators of cell transdifferentiation by comparing diverse transdifferentiation factors ability to bind nucleosomes in vitro. TFs identified by nucleosome binding ability will be compared using biophysical techniques including hydrogen exchange by mass spectrometry and fluorescence resonance energy transfer (FRET). This will give insight into changes in nucleosome structure caused by TF binding. 2) Determine if nucleosome binding capacity is required for TFs transdifferentiation ability. Achievement of the proposed goal will provide insights into the in vivo genomic mechanisms of cellular transdifferentiation and biochemical analysis will provide structural detail of functional domains and activity of pioneer factors, which is a distinguishing feature of my training program.
My proposed graduate research is relevant to public health because my plan to better understand how gene regulatory proteins bind chromosomes in order to change cell fate will improve strategies to develop new cells for therapeutic purposes.