Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark

The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, we have characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies. The results showed that for some H3S10ph antibodies their labeling indeed can be occluded by the concomitant presence of the H3K9me2 mark. Furthermore, we demonstrate that the double H3S10phK9me2 mark is present in pericentric heterochromatin as well as on the fourth chromosome of wild-type polytene chromosomes but not in preparations from JIL-1 or Su(var)3-9 null larvae. Su(var)3-9 is a methyltransferase mediating H3K9 dimethylation. Furthermore, the H3S10phK9me2 labeling overlapped with that of the non-occluded H3S10ph antibodies as well as with H3K9me2 antibody labeling. Interestingly, when a Lac-I-Su(var)3-9 transgene is overexpressed, it upregulates H3K9me2 dimethylation on the chromosome arms creating extensive ectopic H3S10phK9me2 marks suggesting that the H3K9 dimethylation occurred at euchromatic H3S10ph sites. This is further supported by the finding that under these conditions euchromatic H3S10ph labeling by the occluded antibodies was abolished. Thus, our findings indicate a novel role for the JIL-1 kinase in epigenetic regulation of heterochromatin in the context of the chromocenter and fourth chromosome by creating a composite H3S10phK9me2 mark together with the Su(var)3-9 methyltransferase

RNA polymerase II-mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila

JIL-1 is the major kinase controlling the phosphorylation state of histone H3S10 at interphase in Drosophila. In this study, we used three different commercially available histone H3S10 phosphorylation antibodies, as well as an acid-free polytene chromosome squash protocol that preserves the antigenicity of the histone H3S10 phospho-epitope, to examine the role of histone H3S10 phosphorylation in transcription under both heat shock and non-heat shock conditions. We show that there is no redistribution or upregulation of JIL-1 or histone H3S10 phosphorylation at transcriptionally active puffs in such polytene squash preparations after heat shock treatment. Furthermore, we provide evidence that heat shock-induced puffs in JIL-1 null mutant backgrounds are strongly labeled by antibody to the elongating form of RNA polymerase II (Pol IIoser2), indicating that Pol IIoser2 is actively involved in heat shock-induced transcription in the absence of histone H3S10 phosphorylation. This is supported by the finding that there is no change in the levels of Pol IIoser2 in JIL-1 null mutant backgrounds compared with wild type. mRNA from the six genes that encode the major heat shock protein in Drosophila, Hsp70, is transcribed at robust levels in JIL-1 null mutants, as directly demonstrated by qRT-PCR. Taken together, these data are inconsistent with the model that Pol II-dependent transcription at active loci requires JIL-1-mediated histone H3S10 phosphorylation, and instead support a model in which transcriptional defects in the absence of histone H3S10 phosphorylation are a result of structural alterations of chromatin

Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila

Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a `kinase dead\u27 LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the `kinase dead\u27 construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo