Doctor of Philosophy
Phenotypic non-specificity is a phenomenon in which the phenotypes associated with the expression of a given Transcription Factor (TF) are induced or rescued by multiple distinct TFs. Importantly, this phenomenon is observed with TFs from different protein families that recognize distinct DNA binding sites. To further analyze this phenomenon in Drosophila melanogaster, experiments were initiated for the purpose of integrating non-resident TFs into target TF loci via recombinase mediated cassette exchange (subsequent to the introduction of attP sites at the TF loci by CRISPR mediated homology directed repair). Contrary to expectations, no homologous recombinants were identified during the initial CRISPR mediated attempts at gene editing. However, three w+ non-homologous recombinants were identified: two when targeting bcd (Bcd 4 and Bcd 39) and one when targeting Scr (Scr-D1). Bcd 4 and Bcd 39 were the result of transposition of the w67c23 allele into the first intron of the osp gene; whereas Scr-D1 was the result of the insertion of the mini-white gene from the Scr repair template into the genome (with hallmarks of transposition). These non-homologous recombination events suggest that DSBs activate transposable element mobilization. In an alternate approach for studying phenotypic non-specificity, the UAS-GAL4 system was used to express non-resident TFs and assess the functional complementation of loss-of-function alleles at several TF loci. The rescue of six TF loci (lab, Dfd, Scr, Ubx, dsx and fru) was determined using at least 12 non-resident TFs. Five out of the six TF loci were rescued by non-resident TFs: lab was rescued by expression of DSXM; Scr was rescued by expression of FOXO; Ubx was rescued by expression of ANTP and EY; dsx phenotypes were rescued to different extents by the expression of a majority of the non-resident TFs; and fru was rescued by expression of DISCO. In all cases, the rescue was non-uniform across the pleiotropic phenotypes that depend upon the expression of the resident TF. This suggests that the phenomenon of phenotypic non-specificity is differentially pleiotropic.
Summary for Lay Audience
Transcription is the process of copying the DNA sequence of a gene into RNA. “Transcription factors” (TFs) are a class of important proteins that regulate this process by binding to specific DNA sequences adjacent to the gene, thereby turning genes “on” or “off”. Traditionally, each transcription factor is thought to have its own distinct preference with respect to the DNA sequences it binds. Therefore, the function of transcription factors is specific (i.e., that a transcription factor can only regulate a certain number of genes). In my research, I observed multiple occasions of “phenotypic non-specificity” of transcription factors. The results of my research shows that specific transcription factors have the potential to regulate many more distinct genes than expected, and that the function of a transcription factor can be replaced or substituted by another transcription factor. My research indicates that current paradigms of transcription factor function and TF- DNA interaction are not comprehensive and that further studies in this area are needed. Furthermore, I discovered that DNA. damage (DNA double strand breaks) caused by the genetic tool, CRISPR, may destabilize the genome of the organism being manipulated and potentially create unexpected mutations. This discovery should be taken into consideration with regards to the future implementation of CRISPR, especially with respect to clinical trials of CRISPR mediated therapies.
Cheng, Sheng, "Screens for Phenotypic Non-Specificity of Transcription Factor Function in Drosophila melanogaster" (2021). Electronic Thesis and Dissertation Repository. 8303.