Studies of Cys2His2 Zinc Finger Proteins and Their Roles in Biology and Biotechnology.

摘要

Many vital cellular processes are influenced by precise gene expression. From cell cycle progression to cell differentiation, transcription factors (TFs) play key regulatory functions. The Cys2His2 zinc finger motif (ZF) is the most abundant protein domain in the human genome, and is found in over 700 ZF proteins (ZNFs). Many ZNFs are predicted to function as TFs, though only a small fraction have been wholly characterized. By studying ZF domains, we can broaden our knowledge about protein-DNA binding, better our understanding or prediction of the biological function of naturally occurring ZNFs, and utilize this versatile framework to design custom proteins that can target sequences of interest. Here we apply this understanding to explore how changes to the DNA recognition features can be used by us and by nature to alter gene expression.;Due to their stable structure, modularity, and specificity, engineered ZF proteins (ZFPs) have become the basis of a powerful technology. By combining them with different functional domains, they can carry out a variety of cellular activities. In Chapter 2, we describe efforts to improve the use of ZFPs in the context of sequence detection diagnostics by investigating an allosteric regulatory mechanism that utilizes inherent ZF structure changes in bound vs. unbound forms. In Chapter 3 we explore efforts to improve the use of zinc finger nucleases in the context of targeted genome modifications using cell penetrating peptides (CPPs) for protein based live cell delivery. Chapter 4 describes a hypothesis by which natural genetic variation could alter the expression and spectrum of ZNF target genes. By cross-referencing ZNFs with the dbSNP database, we found 65 instances where a single nucleotide polymorphism (SNP) alters critical DNA contacting residues. Two proteins were examined in depth: CTCFL and PRDM10. We analyzed their in vitro binding specificity with Bind-n-Seq, and used RNA-Seq as a functional assay to characterize the consequences of altered DNA specificity on global gene expression and regulation. Although further studies would be necessary, the studies presented here collectively show how a detailed knowledge of C2H2 zinc finger-DNA interactions can improve our understanding of their role in both biology and biotechnology.