Date of Award
Doctor of Philosophy
Theses & dissertations (Interdisciplinary)
Kristen M. Johansen
Epigenetic regulation is an important process utilized by biological systems to control gene expression and organize development, with histone modification enzymes among the most important components in this regulatory network. The predominant interphase H3 Serine10 kinase in Drosophila is JIL-1, which plays an important role in regulating chromosome structure and modulating gene expression. The full-length JIL-1 protein is composed of four domains including a N-terminal domain (NTD), two kinase domains (KDI and KDII), and a unique C-terminal domain (CTD). In order to study the biological importance of these individual domains, a domain analysis research approach was taken and different functions of the JIL-1 kinase were dissected. This thesis will emphasize the significance of the CTD.;From a yeast-two-hybrid screening using the JIL-1 CTD as bait, lamin Dm0 was identified as a potential interaction partner. This interaction was further confirmed by molecular interaction analysis such as pull-down assays and co-immunoprecipitation experiments, and the interface for this interaction was mapped to the predicted globular region within JIL-1 CTD and the C-terminal tail in lamin Dm0. In the JIL-1z2/JIL-1h9 mutant devoid of the lamin interaction domain, we observed a distorted lamina structure in the nurse cells during oogenesis. In addition, genetic analysis in both viability assays and wm4 PEV (positional effect variegation) assays indicates that JIL-1 and lamin Dm0 counteract with each other.;Transgenic analysis using both the CFP-CTD and GFP-Delta-CTD transgenes indicates that the CTD is required for the proper chromosomal localization of JIL-1. Without the CTD, the truncated JIL-1 protein GFP-Delta-CTD loses its affinity for the chromosomes, and mainly localizes to the inter-chromosomal region. I identified a novel H3 binding motif within the CTD that may mediate the association between the CTD and chromatin. Rescue experiments using these transgenes also indicate that the CTD plays an important role in maintaining higher-order chromosome structure with the male X chromosome in particular.;In a PEV system where reporter genes are inserted in pericentric regions, loss of JIL-1 protein results in a dramatic reduction of reporter gene expression. This is consistent with the observation in JIL-1 loss-of-function mutants that the heterochromatin marker H3K9me2 mediated by the histone methyltransferase Su(var)3-9 spreads from the chromocenter to the chromosome arms; however, genetic analysis indicates that JIL-1 and Su(var)3-9 are likely to interact in a novel pathway that is largely independent of HP1. In contrast to the loss-of-function mutants the JIL-1Su(var)3-1 mutant, which carries an early stop codon that removes half of the CTD including both lamin and H3 interaction domains, dramatically increases the expression from the pericentric reporter genes and acts as a gain-of-function mutation in PEV.;In summary, the data above suggest a model in which the CTD of JIL-1 interacts with lamin Dm0 in Drosophila and this interaction is important for the integrity of nuclear lamina. In addition, the CTD can also interact with chromatin and may block the association between lamin and chromatin. The kinase activity of JIL-1 mediates the phosphorylation of H3S10, which can further open the chromatin, allowing the access of RNA Pol II and other transcription machineries and facilitating gene expression.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Bao, Xiaomin, "Functional analysis of the C-terminal domain in the Drosophila JIL-1 histone H3 kinase" (2008). Retrospective Theses and Dissertations. 15794.