RESUMEN
In this issue, Versluis et al.1 use a highly sensitive live-cell imaging system to examine transcription dynamics and functions of various key transcription elongation regulators at the Hsp70 loci.
Asunto(s)
Cromosomas Politénicos , Elongación de la Transcripción Genética , Animales , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Transcripción Genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , ARN Polimerasa II , Transcripción Genética , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Regiones Promotoras Genéticas , Sistemas CRISPR-Cas , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , Regulación de la Expresión Génica , Fosforilación , Unión Proteica , Respuesta al Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
Antibodies to histone modifications and an insulator protein involved in the processes of transcription initiation and elongation are mapped in Drosophila polytene chromosomes. The CHRIZ protein (chromatin insulator) and H3K36me3 histone modification (RNA elongation) are detected only in the localization of housekeeping genes (interbands and gray bands of polytene chromosomes) and never in the regions of developmental genes (black bands and large puffs arising from them). Antibodies to H3S10P histone modification, which is associated with the initial elongation of the RNA strand during transcription, are found exclusively in small puffs, but not in housekeeping gene localization sites or large ecdysone-induced puffs, where housekeeping genes are localized. Antibodies to H4R3me2 histone modification (a co-repressor of the ecdysone receptor) are detected only in large ecdysone-induced puffs.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Genes Esenciales , Histonas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ecdisona , Cromosomas/metabolismo , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , ARN , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Drosophila Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of Drosophila embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of Drosophila. Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.
Asunto(s)
Proteínas de Drosophila , Drosophila , Factor de Transcripción GATA4 , Factor 2 Relacionado con NF-E2 , Activación Transcripcional , Animales , Cromatina/genética , Cromatina/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Cromosomas Politénicos/metabolismo , Xenobióticos/metabolismo , Activación Transcripcional/genéticaRESUMEN
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that modulate multiple aspects of gene activity and RNA processing, including transcription, splicing, localization, translation, and decay of RNA. Interaction of hnRNPs with RNA is a highly dynamic but regulated process. Poly(ADP-ribose) polymerase (PARP)-dependent PARylation of different hnRNPs is a well-known posttranslational modification that affects their interactions with RNA. Here, we described a protocol for in situ localization of RNA-binding proteins (RBPs) on giant polytene chromosomes in Drosophila larval salivary glands, which have been widely used to visualize the dynamic binding profiles of various RBPs and other transcription-related proteins at specific loci on chromosomes. This chapter also includes a stepwise description of RNA:RNA in situ hybridization, in conjunction with immunostaining, using polytene chromosome squashes or intact tissues. We also highlight advanced live cell imaging methods, including FRAP and FLIP, using transgenic lines that express fluorescent-tagged hnRNPs. These cytological approaches can be used to visualize the localization of RNA-binding proteins and their interacting RNAs under different cellular conditions.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , ARN/metabolismoRESUMEN
The reversible posttranslational O-GlcNAc modification of serine or threonine residues of intracellular proteins is involved in many cellular events from signaling cascades to epigenetic and transcriptional regulation. O-GlcNAcylation is a conserved nutrient-dependent process involving two enzymes, with O-GlcNAc transferase (OGT) adding O-GlcNAc and with O-GlcNAcase (OGA) removing it in a manner that's protein- and context-dependent. O-GlcNAcylation is essential for epigenetic regulation of gene expression through its action on Polycomb and Trithorax and COMPASS complexes. However, the important role of O-GlcNAc in adult life and health span has been largely unexplored, mainly due the lack of available model systems. Cataloging the O-GlcNAc proteome has proven useful in understanding the biology of this modification in vivo. In this study, we leveraged a recently developed oga knockout fly mutant to identify the O-GlcNAcylated proteins in adult Drosophilamelanogaster. The adult O-GlcNAc proteome revealed many proteins related to cell and organismal growth, development, differentiation, and epigenetics. We identified many O-GlcNAcylated proteins that play a role in increased growth and decreased longevity, including HCF, SIN3A, LOLA, KISMET, ATX2, SHOT, and FOXO. Interestingly, oga mutant flies are larger and have a shorter life span compared to wild type flies, suggesting increased O-GlcNAc results in increased growth. Our results suggest that O-GlcNAc alters the function of many proteins related to transcription, epigenetic modification and signaling pathways that regulate growth rate and longevity. Therefore, our findings highlight the importance of O-GlcNAc in growth and life span in adult Drosophila.
Asunto(s)
Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Glicoproteínas/metabolismo , Longevidad , Mutación/genética , Proteoma/metabolismo , beta-N-Acetilhexosaminidasas/genética , Animales , Tamaño Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Femenino , Ontología de Genes , N-Metiltransferasa de Histona-Lisina/metabolismo , Masculino , Fenotipo , Cromosomas Politénicos/metabolismo , Alas de Animales/enzimologíaRESUMEN
Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. In this work, we combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Proteínas de Drosophila/genética , Histona Acetiltransferasas/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Epigénesis Genética/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Micronucleoli are among the structures composing the peculiar scenario of the nucleolus in salivary gland nuclei of dipterans representative of Sciaridae. Micronucleolar bodies contain ribosomal DNA and RNA, are transcriptionally active and may appear free in the nucleoplasm or associated with specific chromosome regions in salivary gland nuclei. This report deals with an extreme case of nucleolar fragmentation/dispersion detected in the salivary gland of Schwenkfeldina sp. Such a phenomenon in this species was found to be restricted to cell types undergoing polyteny and seems to be differentially controlled according to the cell type. Furthermore, transcriptional activity was detected in virtually all the micronucleolar bodies generated in the salivary gland. The relative proportion of the rDNA in polytene and diploid tissues showed that rDNA under-replication did not occur in polytene nuclei suggesting that the nucleolar and concomitant rDNA dispersion in Schwenkfeldina sp. may reflect a previously hypothesised process in order to counterbalance the rDNA loss due to the under-replication. The chromosomal distribution of epigenetic markers for the heterochromatin agreed with early cytological observations in this species suggesting that heterochromatin is spread throughout the chromosome length of Schwenkfeldina sp. A comparison made with results from another sciarid species argues for a role played by the heterochromatin in the establishment of the rDNA topology in polytene nuclei of Sciaridae.
Asunto(s)
Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Dípteros/genética , Glándulas Salivales/citología , Animales , Fragmentación del ADN , Replicación del ADN , ADN Ribosómico/metabolismo , Dípteros/metabolismo , Heterocromatina/metabolismo , Cromosomas Politénicos/metabolismo , ARN Ribosómico/metabolismo , Transcripción GenéticaRESUMEN
Changes in the effect of methotrexate (400 µg/kg nutrient medium) combined with high temperature (28°C) of ontogenesis were studied on two hybrid lines of Drosophila melanogaster heterozygous by the vestigial gene that underwent long-term target selection for cutting injury of the wing. Changes in the wing shape, mortality rate, and morphology of polytene chromosomes were studied.
Asunto(s)
Metotrexato/farmacología , Animales , Ontologías Biológicas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Calor , Cromosomas Politénicos/metabolismoRESUMEN
In Drosophila salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations SuURES and Su(var)3-906 results in the polytenization of a substantial fraction of unique and moderately repeated sequences but has almost no effect on satellite DNA replication. The Rap1 interacting factor 1 (Rif) protein is a conserved regulator of replication timing, and in Drosophila, it affects underreplication in polytene chromosomes. We compared the morphology of pericentromeric regions and labeling patterns of in situ hybridization of heterochromatin-specific DNA probes between wild-type salivary gland polytene chromosomes and the chromosomes of Rif1 mutants and SuUR Su(var)3-906 double mutants. We show that, despite general similarities, heterochromatin zones exist that are polytenized only in the Rif1 mutants, and that there are zones that are under specific control of Su(var)3-9. In the Rif1 mutants, we found additional polytenization of the largest blocks of satellite DNA (in particular, satellite 1.688 of chromosome X and simple satellites in chromosomes X and 4) as well as partial polytenization of chromosome Y. Data on pulsed incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into polytene chromosomes indicated that in the Rif1 mutants, just as in the wild type, most of the heterochromatin becomes replicated during the late S phase. Nevertheless, a significantly increased number of heterochromatin replicons was noted. These results suggest that Rif1 regulates the activation probability of heterochromatic origins in the satellite DNA region.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Glándulas Salivales/metabolismo , Animales , Drosophila melanogaster/genética , Mutación/genética , Cromosomas Politénicos/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Proteins Z4/putzig and Chriz/Chromator are involved in the chromatin organization on the promoters of the majority of Drosophila genes. It was shown that the Chriz protein region from aa 273 to 503 is required for the interaction with the Z4 protein. Deletion of this sequence leads to derepression of a number of STAT-dependent genes and development of melanotic tumors in flies. The results of this study suggest that the Chriz protein promotes the recruitment of the Z4 protein to chromatin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción STAT/metabolismo , Animales , Sitios de Unión , Cromatina/química , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/metabolismo , Eliminación de Gen , Cromosomas Politénicos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción STAT/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.
Asunto(s)
Anopheles/genética , Células Germinativas/metabolismo , Malaria/parasitología , Cromosomas Politénicos/metabolismo , Animales , Anopheles/citología , Femenino , Membrana Nuclear/metabolismo , Ovario/citología , Glándulas Salivales/citología , Cromosoma X/metabolismoRESUMEN
TDP-43 is the major disease protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-TDP). Here we identify the transcriptional elongation factor Ell-a shared component of little elongation complex (LEC) and super elongation complex (SEC)-as a strong modifier of TDP-43-mediated neurodegeneration. Our data indicate select targets of LEC and SEC become upregulated in the fly ALS/FTLD-TDP model. Among them, U12 snRNA and a stress-induced long non-coding RNA Hsrω, functionally contribute to TDP-43-mediated degeneration. We extend the findings of Hsrω, which we identify as a chromosomal target of TDP-43, to show that the human orthologue Sat III is elevated in a human cellular disease model and FTLD-TDP patient tissue. We further demonstrate an interaction between TDP-43 and human ELL2 by co-immunoprecipitation from human cells. These findings reveal important roles of Ell-complexes LEC and SEC in TDP-43-associated toxicity, providing potential therapeutic insight for TDP-43-associated neurodegeneration.
Asunto(s)
Proteínas de Unión al ADN/toxicidad , ARN no Traducido/genética , Elongación de la Transcripción Genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Sitios Genéticos , Células HEK293 , Humanos , Masculino , Modelos Biológicos , Proteínas Nucleares/metabolismo , Cromosomas Politénicos/metabolismo , Unión Proteica , ARN Nuclear Pequeño/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/metabolismoRESUMEN
Morphologically, polytene chromosomes of Drosophila melanogaster consist of compact "black" bands alternating with less compact "grey" bands and interbands. We developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, we obtained the replication timetable for all the bands mapped. The results allowed us to compare replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the Drosophila genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in Drosophila.
Asunto(s)
Momento de Replicación del ADN/fisiología , Diploidia , Drosophila melanogaster/genética , Genoma de los Insectos , Cromosomas Politénicos/genética , Animales , Cromosomas de Insectos/química , Cromosomas de Insectos/genética , Cromosomas de Insectos/metabolismo , Replicación del ADN , Mapeo Físico de Cromosoma , Cromosomas Politénicos/metabolismo , Glándulas Salivales/metabolismoRESUMEN
Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding associated with alternation of dense stripes (dark or black bands) and light diffuse areas that encompass alternating less compact gray bands and interbands visible with an electron microscope. In recent years, several approaches have been developed to predict location of morphological structures of polytene chromosomes based on the distribution of proteins on the molecular map of Drosophila genome. Comparison of these structures with the results of analysis of the three-dimensional chromatin organization by the Hi-C method indicates that the morphology of polytene chromosomes represents direct visualization of the interphase nucleus spatial organization into topological domains. Compact black bands correspond to the extended topological domains of inactive chromatin, while interbands are the barriers between the adjacent domains. Here, we discuss the prospects of using polytene chromosomes to study mechanisms of spatial organization of interphase chromosomes, as well as their dynamics and evolution.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Drosophila/genética , Cromosomas Politénicos/metabolismo , Animales , Cromatina/genética , Cromosomas Politénicos/genéticaRESUMEN
BACKGROUND: It is well recognized that the interphase chromatin of higher eukaryotes folds into non-random configurations forming territories within the nucleus. Chromosome territories have biologically significant properties, and understanding how these properties change with time during lifetime of the cell is important. Chromosome-nuclear envelope (Chr-NE) interactions play a role in epigenetic regulation of DNA replication, repair, and transcription. However, their role in maintaining chromosome territories remains unclear. RESULTS: We use coarse-grained molecular dynamics simulations to study the effects of Chr-NE interactions on the dynamics of chromosomes within a model of the Drosophila melanogaster regular (non-polytene) interphase nucleus, on timescales comparable to the duration of interphase. The model simulates the dynamics of chromosomes bounded by the NE. Initially, the chromosomes in the model are prearranged in fractal-like configurations with physical parameters such as nucleus size and chromosome persistence length taken directly from experiment. Time evolution of several key observables that characterize the chromosomes is quantified during each simulation: chromosome territories, chromosome entanglement, compactness, and presence of the Rabl (polarized) chromosome arrangement. We find that Chr-NE interactions help maintain chromosome territories by slowing down and limiting, but not eliminating, chromosome entanglement on biologically relevant timescales. At the same time, Chr-NE interactions have little effect on the Rabl chromosome arrangement as well as on how chromosome compactness changes with time. These results are rationalized by simple dimensionality arguments, robust to model details. All results are robust to the simulated activity of topoisomerase, which may be present in the interphase cell nucleus. CONCLUSIONS: Our study demonstrates that Chr-NE attachments may help maintain chromosome territories, while slowing down and limiting chromosome entanglement on biologically relevant timescales. However, Chr-NE attachments have little effect on chromosome compactness or the Rabl chromosome arrangement.
Asunto(s)
Drosophila melanogaster/genética , Membrana Nuclear/metabolismo , Cromosomas Politénicos/química , Cromosomas Politénicos/metabolismo , Animales , Cromosomas de Insectos/química , Cromosomas de Insectos/genética , Cromosomas de Insectos/metabolismo , Drosophila melanogaster/metabolismo , Interfase , Modelos Moleculares , Cromosomas Politénicos/genéticaRESUMEN
New data on the organization of genes entirely located in the open domains for chromatin transcription and occupying only one chromosome structure (interband) were obtained. The characteristic features of these genes are the small size (on average, 1-2 kb), depletion of the replicative complex proteins in the regulatory region, and the presence of specific motifs for binding transcription factors, as compared to the genes occupying two structures (interband and gray band). The biological function of these genes is associated primarily with the processes of gene expression and RNA metabolism.
Asunto(s)
Cromatina , Proteínas de Unión al ADN , Proteínas de Drosophila , Regulación de la Expresión Génica/fisiología , Cromosomas Politénicos , ARN , Animales , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , ARN/biosíntesis , ARN/genéticaRESUMEN
High-throughput assays of three-dimensional interactions of chromosomes have shed considerable light on the structure of animal chromatin. Despite this progress, the precise physical nature of observed structures and the forces that govern their establishment remain poorly understood. Here we present high resolution Hi-C data from early Drosophila embryos. We demonstrate that boundaries between topological domains of various sizes map to DNA elements that resemble classical insulator elements: short genomic regions sensitive to DNase digestion that are strongly bound by known insulator proteins and are frequently located between divergent promoters. Further, we show a striking correspondence between these elements and the locations of mapped polytene interband regions. We believe it is likely this relationship between insulators, topological boundaries, and polytene interbands extends across the genome, and we therefore propose a model in which decompaction of boundary-insulator-interband regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.
Asunto(s)
Cromatina/metabolismo , Drosophila melanogaster/embriología , Elementos Aisladores , Cromosomas Politénicos/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Histonas/metabolismoRESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Fosfoserina/metabolismo , Elongación de la Transcripción Genética , Animales , Eucromatina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Respuesta al Choque Térmico/genética , Immunoblotting , Inmunohistoquímica , Operón Lac/genética , Mutación/genética , Fosforilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Cromosomas Politénicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , TransgenesRESUMEN
We have developed a procedure that enables visualization of the genomic loci that are bound by complexes formed by a specific combination of chromatin-binding proteins. This procedure is based on imaging bimolecular fluorescence complementation (BiFC) complexes on Drosophila polytene chromosomes. BiFC complexes are formed by the facilitated association of two fluorescent protein fragments that are fused to proteins that interact with, or are in close proximity to, each other. The intensity of BiFC complex fluorescence at individual genomic loci is greatly enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. The loci that are bound by the complexes are mapped by comparing the locations of BiFC complex fluorescence with the stereotypical banding patterns of the chromosomes. We describe strategies for the design, expression, and validation of fusion proteins for the analysis of BiFC complex binding on polytene chromosomes. We detail protocols for the preparation of polytene chromosome spreads that have been optimized for the purpose of BiFC complex visualization. Finally, we provide guidance for the interpretation of results from studies of BiFC complex binding on polytene chromosomes and for comparison of the genomic loci that are bound by BiFC complexes with those that are bound by subunits of the corresponding endogenous complexes. The visualization of BiFC complex binding on polytene chromosomes provides a unique method to visualize multiprotein complex binding at specific loci, throughout the genome, in individual cells.