Host cell factor-1 (HCF-1) is an evolutionarily conserved protein first discovered through its interaction with the herpes simplex viral transactivator VP16. Both the amino-terminal VP 16 interaction domain and the adjacent, less studied basic domain of HCF-1 are essential in cell cycle regulation. However, the mechanism(s) of this regulation is unknown. Our objective was to provide insight into the importance of the basic domain by studying proteins that interact with this region. Using the yeast two-hybrid system with the HCF-1 basic region as bait, we identified a novel protein named HCF-1 interacting zinc finger protein (HIZ). The purpose of this research was to characterize HIZ and determine if its interaction with HCF-1 could reveal a novel cellular target for HCF-1. A putative HIZ full-length sequence was determined and its primary structure was studied in detail. HIZ contains 16 C₂H₂ zinc fingers in its amino terminal. HIZ also contains a novel glutamine-rich motif adjacent to a potent autonomous transactivation domain. The presence of a DNA-bincing domain structure and a strong, functional transactivation domain indicate that HIZ is a novel transcriptional factor. Northern blot analysis showed tissue specific expression of HIZ with highest levels in the testis, skeletal muscle, liver and pancreas, suggesting possible roles in spermatogenesis, differentiation and metabolism. The HIZ-HCF-1 interaction was verified 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 using glutathione-s-transferase (GST) pull-down assays and the minimal HIZ domain for HCF-1 interaction was mapped to the same region critical for transactivation. Recent studies have identified the transcription factors Miz-1, GABP, LZIP, Zhangfei and PGC-1β as proteins that interact with the two domains of HCF-1 important in cell cycle control. Also, these studies have shown that interaction with HCF -1 is important in regulation of their transactivation potential. Thus, the effect of HCF-1 on HIZ activation was studied using transient transfection assays. Similar to its effect on the known cell cycle inhibitor, Miz-1, our studies showed that cotransfection with HCF-1 significantly inhibited HIZ transactivation. The expression pattern of HIZ in a matched tumour/normal tissue array showed that HIZ is expressed at a significantly lower level in tumours of the lung and uterus in comparison to normal tissues from the same individual. This finding suggests a correlation between HIZ expression and tumour formation, possibly in conjunction with the known cell cycle regulator HCF -1. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22680 |
| Date | 03 1900 |
| Creators | Wong, Helen |
| Contributors | Capone, John, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0099 seconds