RESUMO
Heterochromatic loci can exhibit different transcriptional states in genetically identical cells. A popular model posits that the inheritance of modified histones is sufficient for inheritance of the silenced state. However, silencing inheritance requires silencers and therefore cannot be driven by the inheritance of modified histones alone. To address these observations, we determined the chromatin architectures produced by strong and weak silencers in Saccharomyces. Strong silencers recruited Sir proteins and silenced the locus in all cells. Strikingly, weakening these silencers reduced Sir protein recruitment and stably silenced the locus in some cells; however, this silenced state could probabilistically convert to an expressed state that lacked Sir protein recruitment. Additionally, changes in the constellation of silencer-bound proteins or the concentration of a structural Sir protein modulated the probability that a locus exhibited the silenced or expressed state. These findings argued that distinct silencer states generate epigenetic states and regulate their dynamics.
Assuntos
Heterocromatina , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , Inativação Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismoRESUMO
Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. However, a broader hypothesis suggests that chromatin compaction can be both a cause and a consequence of the locus histone modification state, with a tight bidirectional interaction underpinning bistable transcriptional states. To rigorously test this hypothesis, we developed a mathematical model for the dynamics of the HMR locus in Saccharomyces cerevisiae, that incorporates activating histone modifications, silencing proteins, and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states (and vice versa), and protein binding/histone modification levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. Such bidirectional feedback between chromatin compaction and the histone modification state may be a widespread and important regulatory mechanism given the hallmarks of many heterochromatic regions: physical chromatin compaction and dimerizing (or multivalent) silencing proteins.
Assuntos
Cromatina , Proteínas de Saccharomyces cerevisiae , Cromatina/genética , Cromatina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Código das Histonas , Retroalimentação , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Heterochromatic domains are complex structures composed of nucleosome arrays that are bound by silencing factors. This composition raises the possibility that certain configurations of nucleosome arrays facilitate heterochromatic silencing. We tested this possibility in Saccharomyces cerevisiae by systematically altering the distance between heterochromatic nucleosome-depleted regions (NDRs), which is predicted to affect local nucleosome positioning by limiting how nucleosomes can be packed between NDRs. Consistent with this prediction, serial deletions that altered the distance between heterochromatic NDRs revealed a striking oscillatory relationship between inter-NDR distance and defects in nucleosome positioning. Furthermore, conditions that caused poor nucleosome positioning also led to defects in both heterochromatin stability and the ability of cells to generate and inherit epigenetic transcriptional states. These findings strongly suggest that nucleosome positioning can contribute to formation and maintenance of functional heterochromatin and point to previously unappreciated roles of NDR positioning within heterochromatic domains.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Inativação Gênica , Saccharomyces cerevisiae/metabolismoRESUMO
Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. Here, we propose a broader view: chromatin compaction can be both a cause and a consequence of the histone modification state, and this tight bidirectional interaction can underpin bistable transcriptional states. To test this theory, we developed a mathematical model for the dynamics of the HMR locus in S. cerevisiae, that incorporates activating histone modifications, silencing proteins and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states, and vice versa, and protein binding levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. This bidirectional feedback between chromatin compaction and the histone modification state may be an important regulatory mechanism at many loci.
RESUMO
Wolbachia is a globally distributed bacterial endosymbiont present in arthropods and nematodes. The advent of sensitive PCR-based approaches has greatly facilitated the identification of Wolbachia-infected individuals and analysis of population infection levels. Here, a complementary visual fluorescence-based Wolbachia screening approach is described. Through the use of the fluorescent dye Syto-11, Wolbachia can be efficiently detected in various Drosophila tissues, including ovaries. Syto-11 also stains Wolbachia in other insects. Because Wolbachia is inherited through the maternal germ line, bacteria reside in the ovaries of flies in infected populations. An advantage of this staining approach is that it informs about Wolbachia titer as well as its tissue and cellular distribution. Using this method, the infection status of insect populations in two central California locations was determined, and variants with unusually low or high Wolbachia titers were isolated. In addition, a variant with ovarioles containing both infected and uninfected egg chambers was identified. Syto-11 staining of Cardinium- and Spiroplasma-infected insects was also analyzed.
Assuntos
Drosophila melanogaster/microbiologia , Microscopia de Fluorescência/métodos , Simbiose , Wolbachia/isolamento & purificação , Animais , DNA Bacteriano/análise , Células Germinativas/microbiologia , Ácidos Nucleicos/metabolismo , Reação em Cadeia da Polimerase , Spiroplasma/genética , Spiroplasma/isolamento & purificação , Fatores de Tempo , Wolbachia/genéticaRESUMO
Genetic information acquires additional meaning through epigenetic regulation, the process by which genetically identical cells can exhibit heritable differences in gene expression and phenotype. Inheritance of epigenetic information is a critical step in maintaining cellular identity and organismal health. In Saccharomyces cerevisiae, one form of epigenetic regulation is the transcriptional silencing of two mating-type loci, HML and HMR, by the SIR-protein complex. To focus on the epigenetic dimension of this gene regulation, we conducted a forward mutagenesis screen to identify mutants exhibiting an epigenetic or metastable silencing defect. We utilized fluorescent reporters at HML and HMR, and screened yeast colonies for epigenetic silencing defects. We uncovered numerous independent sir1 alleles, a gene known to be required for stable epigenetic inheritance. More interestingly, we recovered a missense mutation within SIR2, which encodes a highly conserved histone deacetylase. In contrast to sir1Δ, which exhibits states that are either fully silenced or fully expressed, this sir2 allele exhibited heritable states that were either fully silenced or expressed at an intermediate level. The heritable nature of this unique silencing defect was influenced by, but not completely dependent on, changes in rDNA copy number. Therefore, this study revealed a heritable state of intermediate silencing and linked this state to a central silencing factor, Sir2.
Assuntos
Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genética , Alelos , Epigênese Genética , Expressão Gênica/genética , Inativação Gênica/fisiologia , Genes Fúngicos Tipo Acasalamento , Característica Quantitativa Herdável , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Telômero/metabolismo , Transcrição Gênica/genéticaRESUMO
Heterochromatic gene silencing is an important form of gene regulation that usually requires specific histone modifications. A popular model posits that inheritance of modified histones, especially in the form of H3-H4 tetramers, underlies inheritance of heterochromatin. Because H3-H4 tetramers are randomly distributed between daughter chromatids during DNA replication, rare occurrences of asymmetric tetramer inheritance within a heterochromatic domain would have the potential to destabilize heterochromatin. This model makes a prediction that shorter heterochromatic domains would experience unbalanced tetramer inheritance more frequently, and thereby be less stable. In contrast to this prediction, we found that shortening a heterochromatic domain in Saccharomyces had no impact on the strength of silencing nor its heritability. Additionally, we found that replisome mutations that disrupt inheritance of H3-H4 tetramers had only minor effects on heterochromatin stability. These findings suggest that histones carry little or no memory of the heterochromatin state through DNA replication.
Assuntos
Epigênese Genética , Histonas/metabolismo , Biopolímeros/química , Biopolímeros/metabolismo , Replicação do DNA , Inativação Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/química , Leveduras/genéticaRESUMO
Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes. Tuning the lipid composition and curvature appropriately demands an appreciation of the native phospholipid and curvature environments that can form transiently in cells. Supported lipid bilayers on glass coverslips that contain phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) make actin bundles termed filopodia-like structures that contain fascin and have vasodilator-stimulated phosphoprotein (VASP) at their growing tips, indicating that these resemble filopodia growing from the plasma membrane. The combination of PI(4,5)P2 and phosphatidylinositol 3-phosphate in curved liposomes or supported bilayers on glass nanospheres uses Snx9, Cdc42, N-WASP (neuronal-Wiskott-Aldrich syndrome protein), and Arp2/3 complex for actin polymerization suggesting that this membrane may mimic the progression from plasma membrane to endosomes. Here we describe how to prepare high-speed supernatant frog egg extracts and phosphoinositide-containing liposomes and supported lipid bilayers that can assemble actin structures. We also describe the methods we use to assay actin polymerization using microscopy and spectrofluorometry and our protocol for immunodepleting specific proteins from extracts.
Assuntos
Actinas/metabolismo , Bicamadas Lipídicas/síntese química , Lipossomos/síntese química , Extratos de Tecidos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Sistema Livre de Células , Feminino , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nanosferas/metabolismo , Óvulo/citologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Polimerização , Pseudópodes , Nexinas de Classificação/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
As early endosomes mature, the SAND-1/CCZ-1 complex acts as a guanine nucleotide exchange factor (GEF) for RAB-7 to promote the activity of its effector, HOPS, which facilitates late endosome-lysosome fusion and the consumption of AP-3-containing vesicles. We show that CCZ-1 and the HOPS complex are essential for the biogenesis of gut granules, cell type-specific, lysosome-related organelles (LROs) that coexist with conventional lysosomes in Caenorhabditis elegans intestinal cells. The HOPS subunit VPS-18 promotes the trafficking of gut granule proteins away from lysosomes and functions downstream of or in parallel to the AP-3 adaptor. CCZ-1 also acts independently of AP-3, and ccz-1 mutants mistraffic gut granule proteins. Our results indicate that SAND-1 does not participate in the formation of gut granules. In the absence of RAB-7 activity, gut granules are generated; however, their size and protein composition are subtly altered. These observations suggest that CCZ-1 acts in partnership with a protein other than SAND-1 as a GEF for an alternate Rab to promote gut granule biogenesis. Point mutations in GLO-1, a Rab32/38-related protein, predicted to increase spontaneous guanine nucleotide exchange, specifically suppress the loss of gut granules by ccz-1 and glo-3 mutants. GLO-3 is known to be required for gut granule formation and has homology to SAND-1/Mon1-related proteins, suggesting that CCZ-1 functions with GLO-3 upstream of the GLO-1 Rab, possibly as a GLO-1 GEF. These results support LRO formation occurring via processes similar to conventional lysosome biogenesis, albeit with key molecular differences.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Grânulos Citoplasmáticos/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endossomos/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/embriologia , Proteínas Mutantes/metabolismo , Transporte Proteico , Supressão Genética , proteínas de unión al GTP Rab7RESUMO
The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(-) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(-) and BLOC-1(-) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans.