Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Kenneth S. Zaret

Abstract

Transcription factors (TFs) forage the genome to instruct cell plasticity, identity, and differentiation. These developmental processes are elicited through TF engagement with chromatin. Yet, how and which TFs can engage with chromatin and thus, nucleosomes, remains largely unexplored. Pioneer TFs are TF that display a high affinity for nucleosomes. Extensive genetic and biochemical studies on the pioneer TF FOXA, a driver of fibroblast to hepatocyte reprogramming, revealed its nucleosome binding ability and chromatin targeting lead to chromatin accessibility and subsequent cooperative binding of TFs. Similarly, a number of reprogramming TFs have been suggested to have pioneering activity due to their ability to target compact chromatin and increase accessibility and enhancer formation in vivo. But whether these factors directly interact with nucleosomes remains to be assessed. Here we test the nucleosome binding ability of the cell reprogramming TFs, Oct4, Sox2, Klf4 and cMyc, that are required for the generation of induced pluripotent stem cells. In addition, we also test neuronal and macrophage reprogramming TFs. Our study shows that reprogramming TFs bind nucleosomes with a range of nucleosome binding affinities, indicating that although specific cocktails of TFs are required for reprogramming, mechanistically these TFs show differential nucleosome interacting behaviors. These results allowed us to assess differential features between TFs nucleosome binding ability and to correlate their binding with reprogramming potential.

To determine how general is nucleosome binding we extended our analysis to screen 593 of the 2,000 predicted human TFs in the genome for potential nucleosome binding and validated their binding in solution. Based on 3D structural analysis, we proposed that strong nucleosome binders anchor DNA through short -helixes and have a flexible and adaptable DNA binding domain while weak nucleosome binders use -sheets or unstructured regions and have a higher rigidity within their DNA binding domain. Through the experiments presented in this dissertation we present the first study revealing the shared structural features contributing to nucleosome binding potential of pioneer TFs and thus allow for predication of novel pioneer TFs with cell reprogramming potential.

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