Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Molecular, Cellular and Developmental Biology
Kristen M. Johansen
In Drosophila, JIL-1 Kinase specifically localizes to euchromatic interband regions of polytene chromosomes and is responsible for histone H3S10 phosphorylation at interphase. Previous genetic interaction assays have demonstrated that the JIL-1 protein can counterbalance the effect of the major heterochromatin components Su(var)3-7 on position-effect variegation (PEV). In this study, we show that the haplo-enhancer effect of JIL-1 has the ability to counterbalance the haplo-suppressor effect of two other major heterochromatin components, Su(var)3-9 and Su(var)2-5, on position-effect variegation, providing evidence that a finely tuned balance between the levels of JIL-1 and the major heterochromatin components contributes to the regulation of gene expression. To test whether this was a causative effect of the epigenetic H3S10 phosphorylation mark, or whether the effect of the JIL-1 protein on PEV was in fact caused by other functions or structural features of the protein, we transgenically expressed various truncated versions of JIL-1, with or without kinase activity, and assessed their effect on PEV and heterochromatic spreading. The results indicate that the gross perturbation of polytene chromosome morphology observed in JIL-1 null mutants is unrelated to gene silencing in PEV and is likely to occur as a result of faulty polytene chromosome alignment and/or organization, separate from epigenetic regulation of chromatin structure.
Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of wm4 PEV depending on the precise levels of JIL-1 kinase activity. To explore the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene locus, we performed ChIP-qPCR analysis. Our results indicate that the H3K9me2 level at the white gene locus directly correlates with its level of expression and that H3K9me2 levels in turn are regulated by H3S10ph.
To further study the localization of H3S10 phosphorylation and H3K9 dimethylation as well as their interplay, genome-wide analysis of JIL-1 kinase binding sites and H3S10ph or H3K9me2 distribution by ChIP-seq was conducted. Furthermore, an analysis of whole genome transcription level changes by RNA-seq in the absence of JIL-1 was used to study the role of this interplay in regulation of gene transcription. The results show that down-regulation of genes in the JIL-1 mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas up-regulation of a gene was correlated with loss of or diminished H3K9me2. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10ph functions to indirectly maintain active transcription by counteracting H3K9me2 and gene silencing.
Wang, Chao, "Epigenetic interplay between histone H3K9me2 and JIL-1 mediated histone H3S10ph" (2014). Graduate Theses and Dissertations. 14241.