The Drosophila hindsight (hnt) gene encodes a C2H2-type zinc-finger (ZNF) protein crucial for epithelial morphogenesis. The human HNT homologue, RREB1, functions as a transcriptional modulator and regulates several tumor suppressor genes. HNT’s functional motifs, target genes and its regulatory abilities have not been elucidated. Here I showed that the C-terminal region of HNT containing the last five of 14 ZNFs (ZNF 10-14) binds in vitro to DNA-elements similar to those identified for RREB-1. I then mapped HNT’s endogenous binding sites on polytene chromosomes and focus on two, at 4C and 60C, which are associated with the hnt and nervy (nvy) loci, respectively. Sequence analysis of the bound fragments shows conservation of motifs similar to those bound by HNT in vitro. Data from both hnt loss- and gain-of-function experiments show that HNT attenuates the transcription of the hnt and nvy genes in several tissues and developmental stages. I show that the identified HNT C-terminal DNA binding domain ZNF 10-14 is not required for these regulatory functions. I further delineate the minimal functional motifs of HNT in transcriptional regulation and show that its ninth ZNF in isolation has a repressive activity and is sufficient to confer many regulatory functions of HNT. On the other hand, mutation of ZNF 9 in the context of the full-length protein indicates that it is not necessary for HNT functions. Interestingly, ZNF 9 has been lost from HNT vertebrate homologues. I propose two redundant mechanisms of transcriptional regulation by HNT: one is mediated by the potential protein-interaction abilities of ZNF 9; another is through cooperation of other ZNF motifs of HNT; the DNA binding abilities conferred by the C-terminal five fingers may be essential for the latter mechanism.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/65485 |
| Date | 19 June 2014 |
| Creators | Ming, Liang |
| Contributors | Lipshitz, Howard |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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