The JIL-1 Kinase Does Not Phosphorylate H3S28 or Recruit 14-3-3 to Active Genes in Drosophila. Chao Wang, Changfu Yao, Yeran Li, Weili Cai, Jack Girton, Jørgen Johansen, Kristen M. Johansen. Biochemistry, Biophysics & Mol Biol, Iowa State University, Ames, IA.

   JIL-1 is the major kinase controlling phosphorylation of H3S10 and functions to counteract heterochromatization and gene silencing (Wang et al, J Cell Sci 124:4309). However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes (Ivaldi et al, Genes Dev 21:2818; Kellner et al, Genome Res 22:1081; Karam et al, PLoS Genetics: e1000975). Since these findings are incompatible with the results of Cai et al (Development 135:2917) demonstrating robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at heat shock-induced polytene chromosome puffs, we reexamined JIL-1's possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in the JIL-1 null mutant at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a LacI-JIL-1 targeting system to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes.Supported by NIH grant GM62916.