RESUMEN
dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.
Asunto(s)
Adenosina Trifosfatasas/genética , Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Nucleosomas/genética , Cromosomas Politénicos/genética , Adenosina Trifosfatasas/metabolismo , Animales , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Interfase/genética , Unión Proteica , Glándulas Salivales/citología , Glándulas Salivales/metabolismo , CohesinasRESUMEN
Although tremendous progress has been made toward identifying factors that regulate nucleosome structure and positioning, the mechanisms that regulate higher-order chromatin structure remain poorly understood. Recent studies suggest that the ISWI chromatin-remodeling factor plays a key role in this process by promoting the assembly of chromatin containing histone H1. To test this hypothesis, we investigated the function of H1 in Drosophila. The association of H1 with salivary gland polytene chromosomes is regulated by a dynamic, ATP-dependent process. Reducing cellular ATP levels triggers the dissociation of H1 from polytene chromosomes and causes chromosome defects similar to those resulting from the loss of ISWI function. H1 knockdown causes even more severe defects in chromosome structure and a reduction in nucleosome repeat length, presumably due to the failure to incorporate H1 during replication-dependent chromatin assembly. Our findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes.
Asunto(s)
Adenosina Trifosfatasas/genética , Cromosomas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Histonas/metabolismo , Factores de Transcripción/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Modificados Genéticamente , Western Blotting , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Cromosomas/genética , Replicación del ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Femenino , Recuperación de Fluorescencia tras Fotoblanqueo , Histonas/genética , Interfase , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Microscopía Confocal , Nucleosomas/genética , Nucleosomas/metabolismo , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , Factores de Transcripción/metabolismoRESUMEN
The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a 2-MDa chromatin-remodeling complex. brm was identified in a screen for transcriptional activators of homeotic genes and subsequently shown to play a global role in transcription by RNA polymerase II. To gain insight into the targeting, function, and regulation of the BRM complex, we screened for mutations that genetically interact with a dominant-negative allele of brm (brm(K804R)). We first screened for dominant mutations that are lethal in combination with a brm(K804R) transgene under control of the brm promoter. In a distinct but related screen, we identified dominant mutations that modify eye defects resulting from expression of brm(K804R) in the eye-antennal imaginal disc. Mutations in three classes of genes were identified in our screens: genes encoding subunits of the BRM complex (brm, moira, and osa), other proteins directly involved in transcription (zerknullt and RpII140), and signaling molecules (Delta and vein). Expression of brm(K804R) in the adult sense organ precursor lineage causes phenotypes similar to those resulting from impaired Delta-Notch signaling. Our results suggest that signaling pathways may regulate the transcription of target genes by regulating the activity of the BRM complex.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Alelos , Animales , Proteínas de Ciclo Celular/genética , Mapeo Cromosómico , Drosophila/genética , Proteínas de Drosophila/genética , Anomalías del Ojo/genética , Anomalías del Ojo/ultraestructura , Técnica del Anticuerpo Fluorescente Indirecta , Prueba de Complementación Genética , Microscopía Electrónica de Rastreo , Receptores Notch/genética , Transactivadores/genética , Transgenes , Cromosoma XRESUMEN
In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Histonas/genética , Animales , Cromatina/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Microscopía Fluorescente/métodosRESUMEN
The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (KIS-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of KIS-L in gene expression, we examined its distribution on larval salivary gland polytene chromosomes. KIS-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of KIS-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that KIS-L facilitates an early step in transcriptional elongation by Pol II.
Asunto(s)
ADN Helicasas/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas de Homeodominio/genética , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Animales , Ensamble y Desensamble de Cromatina/fisiología , ADN Helicasas/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/metabolismoRESUMEN
DEAD-box proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling. Here, we report identification and characterization of the Drosophila protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-box proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY. Mutations in DBY are a frequent cause of male infertility in humans. Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation. Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype. Hypomorphic bel mutants are male-sterile. Bel is also closely related to the Drosophila DEAD-box protein Vasa (Vas), a germ line-specific translational regulator. We find that Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and that bel function is required for female fertility. However, unlike Vas, Bel is not specifically enriched in embryonic pole cells. We conclude that the DEAD-box protein Bel has evolutionarily conserved roles in fertility and development.