Understanding the contribution of individual zinc fingers to a multi-functional, polydactyl transcription factor

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https://ir.uiowa.edu/etd/1290; https://ir.uiowa.edu/cgi/viewcontent.cgi?article=5329&context=etd
Baxley, Ryan M.
The University of Iowa; University of Iowa
Drosophila; Insulator; Oogenesis; Su(Hw); Transcription; Zinc Finger; Cell Biology
thesis / dissertation description
Suppressor of Hairy-wing [Su(Hw)] is a twelve zinc-finger (ZF), DNA binding transcription factor. Su(Hw) has been well characterized as critical component of the gypsy insulator complex, required for the enhancer blocking and the barrier activity of the insulator. In addition to gypsy, Su(Hw) localizes to ~3,000 binding sites in the Drosophila genome, with association to a subset of sites required for female germline development. Loss of Su(Hw) results in activation of a developmental checkpoint and apoptosis at mid- oogenesis, with a critical role during oogenesis in down-regulation of neural genes. Studies of Su(Hw) function have identified transcriptional activator, repressor and insulator roles at distinct binding sites. Current investigations aim to understand the factors that dictate the regulatory output of Su(Hw) at individual sites in the Drosophila genome, with a focus on the ZF domain.A genetic screen was completed to generate novel mutations in su(Hw). After screening more than 8,000 mutagenized chromosomes, we identified four new su(Hw) alleles, including two deletion mutations and two amino acid substitutions disrupting individual ZFs (ZF4 and ZF8). Studies of the ZF4 mutant, Su(Hw)M4M393, revealed that Su(Hw) requires this ZF for female fertility, but notgypsy insulator function. To achieve a comprehensive understanding of the Su(Hw) ZF domain, we generated Su(Hw) mutant proteins carrying disruptions in individual ZFs. Analyses utilizing these proteins have defined the requirement for each ZF in DNA association in vitro. To complement extant ZF alleles, Su(Hw) ZF mutants were expressed in vivo. Analyses of these mutants established how each ZF contributes to SBS occupancy, gypsy insulator function and female fertility. Gene expression and ChIP analyses suggest that some Su(Hw) ZFs may execute roles apart from direct DNA recognition. Genome-wide binding analyses of Su(Hw)M4M393, combined with previous studies, found that the SBS binding motif contains three DNA sequence cores (termed upstream, central and downstream). Analyses of these sequence cores in combination with Su(Hw) ZF mutants have outlined which ZFs associate with each core. Interestingly, the class containing all three sequence cores represents high occupancy SBSs that are enriched for protein factors from functional classes including transcriptional repression, nucleosome remodeling and DNA replication. The class containing the upstream and central core correlates with insulator function, while the class containing the central and downstream cores correlates with activation or repression of Su(Hw) target genes. Finally, in vitro studies of Su(Hw) ZF mutants revealed a DNA bound conformation distinct from wild type Su(Hw).Su(Hw) is a versatile transcription factor able to act as an insulator, activator and repressor. Analyses of SBSs with these functions suggest that DNA sequence, ZF usage, protein partnership and Su(Hw) conformation, combine to dictate regulatory output. Together, these studies provide insight into how discrete ZFs contribute to the roles of a multifunctional, polydactyl transcription factor.