RESUMO
In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1's targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identify interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Interfase , Metilação , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genéticaRESUMO
In Drosophila it has recently been demonstrated that a spindle matrix in the form of a membrane-less macromolecular assembly embeds the microtubule-based spindle apparatus. In addition, two of its constituents, Megator and Chromator, were shown to function as spatial regulators of spindle checkpoint proteins. However, whether the spindle matrix plays a wider functional role in spatially regulating cell cycle progression factors was unknown. Here using a live imaging approach we provide evidence that a number of key cell cycle proteins such as Cyclin B, Polo, and Ran co-localize with the spindle matrix during mitosis. Furthermore, prevention of spindle matrix formation by injection of a function blocking antibody against the spindle matrix protein Chromator results in cell cycle arrest prior to nuclear envelope breakdown. In such embryos the spatial dynamics of Polo and Cyclin B enrichment at the nuclear rim and kinetochores is abrogated and Polo is not imported into the nucleus. This is in contrast to colchicine-arrested embryos where the wild-type dynamics of these proteins are maintained. Taken together these results suggest that spindle matrix formation may be a general requirement for the localization and proper dynamics of cell cycle factors promoting signaling events leading to cell cycle progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Fuso Acromático/metabolismo , Animais , Animais Geneticamente Modificados , Anticorpos/metabolismo , Ciclo Celular/efeitos dos fármacos , Colchicina/farmacologia , Drosophila melanogaster , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Moduladores de Tubulina/farmacologiaRESUMO
A model has been proposed in which JIL-1 kinase-mediated H3S10 and H2Av phosphorylation is required for transcriptional elongation and heat shock-induced chromatin decondensation. However, here we show that although H3S10 phosphorylation is indeed compromised in the H2Av null mutant, chromatin decondensation at heat shock loci is unaffected in the absence of JIL-1 as well as of H2Av and that there is no discernable decrease in the elongating form of RNA polymerase II in either mutant. Furthermore, mRNA for the major heat shock protein Hsp70 is transcribed at robust levels in both H2Av and JIL-1 null mutants. Using a different chromatin remodeling paradigm that is JIL-1 dependent, we provide evidence that ectopic tethering of JIL-1 and subsequent H3S10 phosphorylation recruits PARP-1 to the remodeling site independently of H2Av phosphorylation. These data strongly suggest that H2Av or H3S10 phosphorylation by JIL-1 is not required for chromatin decondensation or transcriptional elongation in Drosophila.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Fosfosserina/metabolismo , Elongação da Transcrição Genética , Animais , Eucromatina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico/genética , Immunoblotting , Imuno-Histoquímica , Óperon Lac/genética , Mutação/genética , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Cromossomos Politênicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , TransgenesRESUMO
In this study we provide evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor/dASCIZ. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. We also confirm a previously documented function of Digitor/dASCIZ as a regulator of Dynein light chain/Cut up expression. Using transgenic expression of a mCitrine-labeled Digitor construct, we show that Digitor/dASCIZ is a nuclear protein that is localized to interband and developmental puff chromosomal regions during interphase but redistributes to the spindle region during mitosis. Its mitotic localization and physical interaction with Skeletor suggest the possibility that Digitor/dASCIZ plays a direct role in mitotic progression as a member of the spindle matrix complex. Furthermore, we have characterized a P-element insertion that is likely to be a true null Digitor/dASCIZ allele resulting in complete pupal lethality when homozygous, indicating that Digitor/dASCIZ is an essential gene. Phenotypic analysis of the mutant provided evidence that Digitor/dASCIZ plays critical roles in regulation of metamorphosis and organogenesis as well as in the DNA damage response. In the Digitor/dASCIZ null mutant larvae there was greatly elevated levels of γH2Av, indicating accumulation of DNA double-strand breaks. Furthermore, reduced levels of Digitor/dASCIZ decreased the resistance to paraquat-induced oxidative stress resulting in increased mortality in a stress test paradigm. We show that an early developmental consequence of the absence of Digitor/dASCIZ is reduced third instar larval brain size although overall larval development appeared otherwise normal at this stage. While Digitor/dASCIZ mutant larvae initiate pupation, all mutant pupae failed to eclose and exhibited various defects in metamorphosis such as impaired differentiation, incomplete disc eversion, and faulty apoptosis. Altogether we provide evidence that Digitor/dASCIZ is a nuclear protein that performs multiple roles in Drosophila larval and pupal development.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição/metabolismo , Animais , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Dano ao DNA , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Expressão Gênica , Genótipo , Resposta ao Choque Térmico , Humanos , Interfase , Metamorfose Biológica , Mitose , Mutação , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição/química , Transcrição GênicaRESUMO
The chromodomain protein, Chromator, is localized to chromosomes during interphase; however, during cell division together with other nuclear proteins Chromator redistributes to form a macro molecular spindle matrix complex that embeds the microtubule spindle apparatus. It has been demonstrated that the CTD of Chromator is sufficient for localization to the spindle matrix and that expression of this domain alone could partially rescue Chro mutant microtubule spindle defects. Furthermore, the presence of frayed and unstable microtubule spindles during mitosis after Chromator RNAi depletion in S2 cells indicated that Chromator may interact with microtubules. In this study using a variety of biochemical assays we have tested this hypothesis and show that Chromator not only has binding activity to microtubules with a Kd of 0.23 µM but also to free tubulin. Furthermore, we have mapped the interaction with microtubules to a relatively small stretch of 139 amino acids in the carboxy-terminal region of Chromator. This sequence is likely to contain a novel microtubule binding interface since database searches did not find any sequence matches with known microtubule binding motifs.
Assuntos
Proteínas de Drosophila/metabolismo , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Anticorpos/imunologia , Proteínas de Drosophila/química , Proteínas de Drosophila/imunologia , Drosophila melanogaster/metabolismo , Imunoprecipitação , Microtúbulos/química , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/imunologiaRESUMO
In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Epigênese Genética , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Mapeamento Cromossômico , Drosophila melanogaster/metabolismo , Feminino , Genoma de Inseto , Larva/genética , Larva/metabolismo , Masculino , Metilação , Fosforilação , Transporte Proteico , Glândulas Salivares/metabolismo , TranscriptomaRESUMO
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.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigênese Genética , Marcadores Genéticos , Heterocromatina/química , Metilação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Epigênese Genética/fisiologia , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , Mutação , Fosforilação/fisiologia , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Serina/genética , Serina/metabolismoRESUMO
JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1's possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that in Drosophila the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a lacO repeat tethering system to target LacI-JIL-1 to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas 14-3-3/análise , Proteínas 14-3-3/genética , Animais , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Deleção de Genes , Genes de Insetos , Histonas/genética , Fosforilação , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Regulação para CimaRESUMO
In this study we have taken advantage of recent whole genome sequencing studies that have determined the DNA content in the heterochromatic regions of each Drosophila chromosome to directly correlate the effect on position-effect variegation of a pericentric insertion reporter line, 118E-10 with the total amount of heterochromatic DNA. Heterochromatic DNA levels were manipulated by adding or subtracting a Y chromosome as well as by the difference in the amount of pericentric heterochromatin between the X and Y chromosome. The results showed a direct, linear relationship between the amount of heterochromatic DNA in the genome and the expression of the w marker gene in the 118E-10 pericentric reporter line and that increasing amounts of heterochromatic DNA resulted in increasing amounts of pigment/gene activity. In Drosophila heterochromatic spreading and gene silencing is counteracted by H3S10 phosphorylation by the JIL-1 kinase, and we further demonstrate that the haplo-enhancer effect of JIL-1 is proportional to the amount of total heterochomatin, suggesting that JIL-1's activity is dynamically modulated to achieve a more or less constant balance depending on the levels of heterochromatic factors present.
Assuntos
Efeitos da Posição Cromossômica , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Genoma de Inseto , Heterocromatina , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Cromossomos de Insetos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Masculino , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. We show that this "internal" matrix is a distinct structure from the microtubule spindle and from a lamin B-containing spindle envelope. By injection of 2000-kDa dextran, we show that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as "struts" stretching the spindle matrix. In addition, we demonstrate that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division.
Assuntos
Proteínas de Drosophila/metabolismo , Microtúbulos/metabolismo , Mitose , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Animais , Animais Geneticamente Modificados , Cromossomos de Insetos/metabolismo , Dextranos/química , Dextranos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Imagem com Lapso de Tempo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteína Vermelha FluorescenteRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Efeitos da Posição Cromossômica , Proteínas de Drosophila/metabolismo , Drosophila/enzimologia , Proteínas do Olho/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Imunoprecipitação da Cromatina , Drosophila/genética , Regulação da Expressão Gênica , Masculino , FosforilaçãoRESUMO
The chromodomain protein, Chromator, can be divided into two main domains, a NH(2)-terminal domain (NTD) containing the chromodomain (ChD) and a COOH-terminal domain (CTD) containing a nuclear localization signal. During interphase Chromator is localized to chromosomes; however, during cell division Chromator redistributes to form a macro molecular spindle matrix complex together with other nuclear proteins that contribute to microtubule spindle dynamics and proper chromosome segregation during mitosis. It has previously been demonstrated that the CTD is sufficient for targeting Chromator to the spindle matrix. In this study, we show that the NTD domain of Chromator is required for proper localization to chromatin during interphase and that chromosome morphology defects observed in Chromator hypomorphic mutant backgrounds can be largely rescued by expression of this domain. Furthermore, we show that the ChD domain can interact with histone H1 and that this interaction is necessary for correct chromatin targeting. Nonetheless, that localization to chromatin still occurs in the absence of the ChD indicates that Chromator possesses a second mechanism for chromatin association and we provide evidence that this association is mediated by other sequences residing in the NTD. Taken together these findings suggest that Chromator's chromatin functions are largely governed by the NH(2)-terminal domain whereas functions related to mitosis are mediated mainly by COOH-terminal sequences.
Assuntos
Cromatina/química , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Interfase/fisiologia , Modelos Moleculares , Proteínas Associadas à Matriz Nuclear/metabolismo , Animais , Drosophila melanogaster/genética , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/genética , Immunoblotting , Imunoprecipitação , Estrutura Terciária de ProteínaRESUMO
Polycomb group (PcG) proteins maintain the spatial expression patterns of genes that are involved in cell-fate specification along the anterior-posterior (A/P) axis. This repression requires cis-acting silencers, which are called PcG response elements (PREs). One of the PcG proteins, Pleiohomeotic (Pho), which has a zinc finger DNA binding protein, plays a critical role in recruiting other PcG proteins to bind to PREs. In this study, we characterized the effects of a pho mutation on embryonic segmentation. pho maternal mutant embryos showed various segmental defects including pair-rule gene mutant patterns. Our results indicated that engrailed and even-skipped genes were misexpressed in pho mutant embryos, which caused embryonic segment defects.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Blastoderma/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Larva/anatomia & histologia , Larva/metabolismo , Mutação , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
In this study, we show that the haplo-enhancer effect of JIL-1 has the ability to counterbalance the haplo-suppressor effect of both Su(var)3-9 and Su(var)2-5 on position-effect variegation, providing evidence that a finely tuned balance between the levels of JIL-1 and the major heterochromatin components contributes to the regulation of gene expression.
Assuntos
Efeitos da Posição Cromossômica , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Alelos , Animais , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Eucromatina/química , Eucromatina/genética , Heterocromatina/química , Heterocromatina/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismoRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Mitose/fisiologia , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Animais , Evolução Biológica , Humanos , Fuso Acromático/químicaRESUMO
The JIL-1 kinase localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays with strong JIL-1 hypomorphic loss-of-function alleles have demonstrated that the JIL-1 protein can counterbalance the effect of the major heterochromatin components on position-effect variegation (PEV) and gene silencing. However, it is unclear whether this was a causative effect of the epigenetic H3S10 phosphorylation mark, or whether the effect of the JIL-1 protein on PEV was in fact caused by other functions or structural features of the protein. By transgenically expressing various truncated versions of JIL-1, with or without kinase activity, and assessing their effect on PEV and heterochromatic spreading, we show that the gross perturbation of polytene chromosome morphology observed in JIL-1 null mutants is unrelated to gene silencing in PEV and is likely to occur as a result of faulty polytene chromosome alignment and/or organization, separate from epigenetic regulation of chromatin structure. Furthermore, the findings provide evidence that the epigenetic H3S10 phosphorylation mark itself is necessary for preventing the observed heterochromatic spreading independently of any structural contributions from the JIL-1 protein.
Assuntos
Efeitos da Posição Cromossômica , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epigênese Genética , Inativação Gênica , Proteínas Serina-Treonina Quinases/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/metabolismo , Olho/anatomia & histologia , Heterocromatina/metabolismo , Histonas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , TransgenesRESUMO
The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms was unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity.
Assuntos
Efeitos da Posição Cromossômica , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Epistasia Genética , Proteínas Serina-Treonina Quinases/genética , Animais , Animais Geneticamente Modificados , Cromossomos de Insetos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Imuno-Histoquímica , Lisina/metabolismo , Masculino , Metilação , Metiltransferases/genética , Microscopia de Fluorescência , Mutação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae. In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies. The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins. Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.
Assuntos
Anticorpos/química , Cromossomos/química , Drosophila/genética , Técnicas Imunoenzimáticas/métodos , Animais , Proteínas de Drosophila/imunologia , Epigênese Genética , Proteínas Serina-Treonina Quinases/imunologiaRESUMO
We have used a yeast two-hybrid interaction assay to identify Asator, a tau-tubulin kinase homolog in Drosophila that interacts directly with the spindle matrix protein Megator. Using immunocytochemical labeling by an Asator-specific mAb as well as by transgenic expression of a GFP-labeled Asator construct, we show that Asator is localized to the cytoplasm during interphase but redistributes to the spindle region during mitosis. Determination of transcript levels using qRT-PCR suggested that Asator is expressed throughout development but at relatively low levels. By P-element excision, we generated a null or strong hypomorphic Asator(exc) allele that resulted in complete adult lethality when homozygous, indicating that Asator is an essential gene. That the observed lethality was caused by impaired Asator function was further supported by the partial restoration of viability by transgenic expression of Asator-GFP in the Asator(exc) homozygous mutant background. The finding that Asator localizes to the spindle region during mitosis and directly can interact with Megator suggests that its kinase activity may be involved in regulating microtubule dynamics and microtubule spindle function.