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.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w(m4) PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w(m4) backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w^m4 PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w^m4 backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine 10 Phosphorylation and Chromatin Remodeling in Vivo

The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH₂-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH₂-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH₂-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein.     

Comprehensive Histone Phosphorylation Analysis and Identification of Pf14-3-3 Protein as a Histone H3 Phosphorylation Reader in Malaria Parasites

The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.     

The Chriz–Z4 complex recruits JIL-1 to polytene chromosomes,a requirement for interband-specific phosphorylation of H3S10

The conserved band-interband pattern is thought to reflect the looped-domain organization of insect polytene chromosomes. Previously, we have shown that the chromodomain protein Chriz and the zinc-finger protein Z4 are essentially required for the maintenance of polytene chromosome structure. Here we show that both proteins form a complex that recruits the JIL-1 kinase to polytene chromosomes, enabling local H3S10 phosphorylation of interband nucleosomal histones. Interband targeting domains were identified at the N-terminal regions of Chriz and Z4, and our data suggest partial cooperation of the complex with the BEAF boundary element protein in polytene and diploid cells. Reducing the core component Chriz by RNAi results in destabilization of the complex and a strong reduction of interband-specific histone H3S10 phosphorylation.     

The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin

The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.     

The JIL-1 kinase regulates the structure of Drosophila polytene chromosomes

The JIL-1 kinase localizes to interband regions of Drosophila polytene chromosomes and phosphorylates histone H3 Ser10. Analysis of JIL-1 hypomorphic alleles demonstrated that reduced levels of JIL-1 protein lead to global changes in polytene chromatin structure. Here we have performed a detailed ultrastructural and cytological analysis of the defects in JIL-1 mutant chromosomes. We show that all autosomes and the female X chromosome are similarly affected, whereas the defects in the male X chromosome are qualitatively different. In polytene autosomes, loss of JIL-1 leads to misalignment of interband chromatin fibrils and to increased ectopic contacts between nonhomologous regions. Furthermore, there is an abnormal coiling of the chromosomes with an intermixing of euchromatic regions and the compacted chromatin characteristic of banded regions. In contrast, coiling of the male X polytene chromosome was not observed. Instead, the shortening of the male X chromosome appeared to be caused by increased dispersal of the chromatin into a diffuse network without any discernable banded regions. To account for the observed phenotypes we propose a model in which JIL-1 functions to establish or maintain the parallel alignment of interband chromosome fibrils as well as to repress the formation of contacts and intermingling of nonhomologous chromatid regions. Electronic Supplementary Material Supplementary material is available for this article at and accessible for authorised users     

The RRM protein NonA from Drosophila forms a complex with the RRM proteins Hrb87F and S5 and the Zn finger protein PEP on hnRNA

The RRM protein NonA, an ubiquitous nuclear protein present in puffs on polytene chromosomes, has been immunopurified as a RNA-protein complex from Drosophila Kc cells. Three other proteins present in the complex have been identified: X4/PEP (protein on ecdysone puffs), a 100-kDa zinc finger RNA-binding protein; the 70-kDa S5 protein, an as yet uncharacterized RNA-binding protein; and P11/Hrb87F, a 38-kDa RRM protein homologous to hnRNP protein A1 from mammals. Monoclonal antibodies against any of the protein components coprecipitate all four proteins although at different ratios. NonA does not coprecipitate with the hrp40 hnRNP proteins and immunolocalizes in a pattern distinct of major hnRNP proteins. Like NonA, X4/PEP, S5, and P11/Hrb87F are present on active sites on polytene chromosomes. The precipitated NonA complex is enriched for certain protein encoding RNAs, notably, histone H3 and H4 RNA.     

The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila

In this study, we show that a reduction in the levels of the JIL-1 histone H3S10 kinase results in the spreading of the major heterochromatin markers dimethyl H3K9 and HP1 to ectopic locations on the chromosome arms, with the most pronounced increase on the X chromosomes. Genetic interaction assays demonstrated that JIL-1 functions in vivo in a pathway that includes Su(var)3-9, which is a major catalyst for dimethylation of the histone H3K9 residue, HP1 recruitment, and the formation of silenced heterochromatin. We further provide evidence that JIL-1 activity and localization are not affected by the absence of Su(var)3-9 activity, suggesting that JIL-1 is upstream of Su(var)3-9 in the pathway. Based on these findings, we propose a model where JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9-mediated heterochromatization.     

The RRM Protein NonA from Drosophila Forms a Complex with the RRM Proteins Hrb87F and S5 and the Zn Finger Protein PEP on hnRNA

The RRM protein NonA, an ubiquitous nuclear protein present in puffs on polytene chromosomes, has been immunopurified as a RNA–protein complex from Drosophila Kc cells. Three other proteins present in the complex have been identified: X4/PEP (protein on ecdysone puffs), a 100-kDa zinc finger RNA-binding protein; the 70-kDa S5 protein, an as yet uncharacterized RNA-binding protein; and P11/Hrb87F, a 38-kDa RRM protein homologous to hnRNP protein A1 from mammals. Monoclonal antibodies against any of the protein components coprecipitate all four proteins although at different ratios. NonA does not coprecipitate with the hrp40 hnRNP proteins and immunolocalizes in a pattern distinct of major hnRNP proteins. Like NonA, X4/PEP, S5, and P11/Hrb87F are present on active sites on polytene chromosomes. The precipitated NonA complex is enriched for certain protein encoding RNAs, notably, histone H3 and H4 RNA.     

Aurora kinase is required for chromosome segregation in tobacco BY-2 cells

Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition.     

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' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' 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.     

Genetic and Molecular Analysis of the X Chromosomal Region 14b17-14c4 in Drosophila Melanogaster: Loss of Function in Nona, a Nuclear Protein Common to Many Cell Types, Results in Specific Physiological and Behavioral Defects

We have performed a genetic analysis of the 14C region of the X chromosome of Drosophila melanogaster to isolate loss of function alleles of no-on-transient A (nonA; 14C1-2; 1-52.3). NONA is a nuclear protein common to many cell types, which is present in many puffs on polytene chromosomes. Sequence data suggest that the protein contains a pair of RNA binding motifs (RRM) found in many single-strand nucleic acid binding proteins. Hypomorphic alleles of this gene, which lead to aberrant visual and courtship song behavior, still contain normally distributed nonA RNA and NONA protein in embryos, and in all available alleles NONA protein is present in puffs of third instar larval polytene chromosomes. We find that complete loss of this general nuclear protein is semilethal in hemizygous males and homozygous cell lethal in the female germline. Surviving males show more extreme defects in nervous system function than have been described for the hypomorphic alleles. Five other essential genes that reside within this region have been partially characterized.