Kristen M. Johansen

The JIL-1 Tandem Kinase Mediates Histone H3 Phosphorylation and Is Required for Maintenance of Chromatin Structure in Drosophila

To analyze the function of the chromosomal kinase JIL-1, we generated an allelic series of hypomorphic and null mutations. JIL-1 is an essential kinase for viability, and reduced levels of JIL-1 kinase activity lead to a global change in chromatin structure. In JIL-1 hypomorphs, euchromatic regions of polytene chromosomes are severely reduced and the chromosome arms condensed. This is correlated with decreased levels of histone H3 Ser10 phosphorylation. These levels can be restored by a JIL-1 transgene placing JIL-1 directly in the pathway mediating histone H3 phosphorylation. We propose a model where JIL-1 kinase activity is required for maintaining the structure of the more open chromatin regions that facilitate gene transcription.

JIL-1: A Novel Chromosomal Tandem Kinase Implicated in Transcriptional Regulation in Drosophila

We have cloned and characterized JIL-1, a novel tandem kinase in Drosophila that associates with the chromosomes throughout the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost 2-fold on the hypertranscribed male X chromosome compared to autosomes. Phylogenetic analysis suggests that JIL-1 together with human MSKs defines a separate family of tandem kinases. That JIL-1 is a functional kinase was demonstrated by autophosphorylation and phosphorylation of histone H3 in vitro. Based on these findings, we propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure.

Regulation of chromatin structure by histone H3S10 phosphorylation

The epigenetic phospho-serine 10 modification of histone H3 has been a puzzle due to its association with two apparently opposed chromatin states. It is found at elevated levels on the highly condensed, transcriptionally inactive mitotic chromosomes yet is also correlated with the more extended chromatin configuration of active genes, euchromatic interband regions, and activated heat shock puffs of Drosophila polytene chromosomes. In addition, phosphorylation of histone H3S10 is up-regulated on the hypertranscribed male X chromosome. Here we review the cellular effects of histone H3S10 phosphorylation and discuss a model for its involvement in regulating chromatin organization and heterochromatization that would be applicable to both interphase and mitotic chromosomes.

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.

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle.

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.

Cell and Molecular Biology of the Spindle Matrix

The concept of a spindle matrix has long been proposed to account for incompletely understood features of microtubule spindle dynamics and force production during mitosis. In its simplest formulation, the spindle matrix is hypothesized to provide a stationary or elastic molecular matrix that can provide a substrate for motor molecules to interact with during microtubule sliding and which can stabilize the spindle during force production. Although this is an attractive concept with the potential to greatly simplify current models of microtubule spindle behavior, definitive evidence for the molecular nature of a spindle matrix or for its direct role in microtubule spindle function has been lagging. However, as reviewed here multiple studies spanning the evolutionary spectrum from lower eukaryotes to vertebrates have provided new and intriguing evidence that a spindle matrix may be a general feature of 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.