Jack Girton

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.

Chromatin structure and the regulation of gene expression: the lessons of PEV in Drosophila.

Position-effect variegation (PEV) was discovered in 1930 in a study of X-ray-induced chromosomal rearrangements. Rearrangements that place euchromatic genes adjacent to a region of centromeric heterochromatin give a variegated phenotype that results from the inactivation of genes by heterochromatin spreading from the breakpoint. PEV can also result from P element insertions that place euchromatic genes into heterochromatic regions and rearrangements that position euchromatic chromosomal regions into heterochromatic nuclear compartments. More than 75 years of studies of PEV have revealed that PEV is a complex phenomenon that results from fundamental differences in the structure and function of heterochromatin and euchromatin with respect to gene expression. Molecular analysis of PEV began with the discovery that PEV phenotypes are altered by suppressor and enhancer mutations of a large number of modifier genes whose products are structural components of heterochromatin, enzymes that modify heterochromatic proteins, or are nuclear structural components. Analysis of these gene products has led to our current understanding that formation of heterochromatin involves specific modifications of histones leading to the binding of particular sets of heterochromatic proteins, and that this process may be the mechanism for repressing gene expression in PEV. Other modifier genes produce products whose function is part of an active mechanism of generation of euchromatin that resists heterochromatization. Current studies of PEV are focusing on defining the complex patterns of modifier gene activity and the sequence of events that leads to the dynamic interplay between heterochromatin and euchromatin.

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.

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor

We have used a yeast two‐hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co‐localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle‐like structure. Deletion construct analysis in S2 cells showed that the COOH‐terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P‐element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

The chromodomain protein, Chromator, interacts with JIL-1 kinase and regulates the structure of Drosophila polytene chromosomes

In this study we have generated two new hypomorphic Chro alleles and analyzed the consequences of reduced Chromator protein function on polytene chromosome structure. We show that in Chro71/Chro612 mutants the polytene chromosome arms were coiled and compacted with a disruption and misalignment of band and interband regions and with numerous ectopic contacts connecting non-homologous regions. Furthermore, we demonstrate that Chromator co-localizes with the JIL-1 kinase at polytene interband regions and that the two proteins interact within the same protein complex. That both proteins are necessary and may function together is supported by the finding that a concomitant reduction in JIL-1 and Chromator function synergistically reduces viability during development. Overlay assays and deletion construct analysis suggested that the interaction between JIL-1 and Chromator is direct and that it is mediated by sequences in the C-terminal domain of Chromator and by the acidic region within the C-terminal domain of JIL-1. Taken together these findings indicate that Chromator and JIL-1 interact in an interband-specific complex that functions to establish or maintain polytene chromosome structure in Drosophila.

Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

The idea of a spindle matrix has long been proposed in order to account for poorly understood features of mitosis. However, its molecular nature and structural composition have remained elusive. Here, we propose that the spindle matrix may be constituted by mainly nuclear-derived proteins that reorganize during the cell cycle to form an elastic gel-like matrix. We discuss this hypothesis in the context of recent observations from phylogenetically diverse organisms that nuclear envelope and intranuclear proteins form a highly dynamic and malleable structure that contributes to mitotic spindle function. We suggest that the viscoelastic properties of such a matrix may constrain spindle length while at the same time facilitating microtubule growth and dynamics as well as chromosome movement. A corollary to this hypothesis is that a key determinant of spindle size may be the amount of nuclear proteins available to form the spindle matrix. Such a matrix could also serve as a spatial regulator of spindle assembly checkpoint proteins during open and semi-open mitosis.

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.

EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry to demonstrate that the EAST protein in Drosophila, which forms an expandable nuclear endoskeleton at interphase, redistributes during mitosis to colocalize with the spindle matrix proteins, Megator and Skeletor. EAST and Megator also colocalize to the intranuclear space surrounding the chromosomes at interphase. EAST is a novel protein that does not have any previously characterized motifs or functional domains. However, we show by immunoprecipitation experiments that EAST is likely to molecularly interact with Megator which has a large NH2‐terminal coiled‐coil domain with the capacity for self assembly. On the basis of these findings, we propose that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis.

Loss-of-function alleles of the JIL-1 histone H3S10 kinase enhance position-effect variegation at pericentric sites in Drosophila heterochromatin.

In this study we show that loss-of-function alleles of the JIL-1 histone H3S10 kinase act as enhancers of position-effect variegation at pericentric sites whereas the gain-of-function JIL-1Su(var)3-1[3] allele acts as a suppressor strongly supporting a functional role for JIL-1 in maintaining euchromatic chromatin and counteracting heterochromatic spreading and gene silencing.

Polytene chromosome squash methods for studying transcription and epigenetic chromatin modification in Drosophila using antibodies.

The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure.