Abstract
JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. 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. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have 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 in Drosophila 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 during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background 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 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only 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 lacO repeat tethering system to target LacI-JIL-1 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.
Highlights
The JIL-1 kinase localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila [1,2]
JIL-1 is not a Histone H3S28 Kinase in Drosophila A recent study by Kellner et al [11] using immunocytochemistry claimed that the H3S28ph mark is present at interbands of polytene chromosomes, that it accumulates at heat shock puffs, and that the JIL-1 kinase is responsible for this H3S28 phosphorylation
To test whether this is the case we performed immunolabeling of polytene squash preparations from wild-type and JIL-1 null third instar larvae using two different commercially available H3S28ph antibodies from Upstate/Millipore (UP) and Cell Signaling (CS), respectively
Summary
The JIL-1 kinase localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila [1,2]. Wang et al [4] provided evidence that gene expression levels at the white locus were directly correlated with the levels of the H3K9me mark independently of the state of the H3S10ph mark, which was not required for either transcription or gene activation to occur These findings taken together with previous studies suggested a model where H3S10 phosphorylation functions to indirectly regulate transcription by counteracting H3K9 dimethylation and gene silencing in a finely tuned balance [3,4,5,6,7,8]. The results suggest that JIL-1 is not a H3S28 kinase and that it is not involved in 14-3-3 recruitment in Drosophila
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