The higher order organization of chromatin within the cell nucleus plays an essential role in epigenetic regulation of gene transcription. In Drosophila, the JIL-1 histone H3S10 kinase has been identified as a major regulator of chromatin structure. JIL-1 associates with chromosomes throughout the cell cycle, and predominantly localizes to less-condensed interband regions on the salivary gland polytene chromosome. In males, JIL-1 is upregulated on the male X chromosome and is associated with MSL dosage compensation complex. Absence of JIL-1 results in loss of interphase H3S10 phosphorylation as well as lethality and multiple defects including severely disrupted polytene chromosome morphology.;Further electron-microscopy analyses of the abnormal polytene chromosome revealed multiple ultrastructural defects including misalignment of interband chromatin fibrils, intermixing of decondensed and compacted chromatin regions, and increased ectopic contacts. These phenotypes suggest an enhanced level of heterochromatin, which was supported by the discovery of up-regulated heterochromatin markers H3K9 dimethylation and HP1 on chromosome arms in JIL-1 mutants. Moreover, the lethality and chromosome morphology defects associated with loss of JIL-1 can be largely rescued by introduction of mutations for heterochromatin components including H3K9 histone methytransferase Su(var)3-9 and Zn-finger protein Su(var)3-7, however not by reduction of HP1, indicating that JIL-1 is required for maintaining euchromatin architecture and normal gene expression via antagonizing ectopic heterochromatization correlated with Su(var)3-9 and Su(var)3-7 while independent of HP1. Furthermore, PEV (position effect variegation) assays using alleles of JIL-1 and Su(var)3-7 revealed the counterbalance between euchromatin and heterochromatin. As a more direct test of our hypothesis, JIL-1 was ectopically targeted to a polytene band using a LacI/lacO tethering system. The JIL-1-mediated ectopic H3S10 phosphorylation was found sufficient to induce a change in higher order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state, while this effect was absent when a "kinase dead" JIL-1 without H3S10 phosphorylation activity was tethered. This finding provides direct evidence that a histone modification might function as a causative regulator of higher order chromatin structure in vivo.