RESUMO
Drosophila imaginal discs often change their cell fate under stress conditions, and this phenomenon, called transdetermination (TD), has long been a useful model for studying cell fate plasticity during regeneration. We previously identified a chromatin-associated protein, Winged Eye (Wge), which induces eye-to-wing TD upon its over-expression in eye imaginal discs. However, the molecular mechanism of Wge-mediated TD remains obscure. Here, we analyzed Wge-interacting proteins and found that several heterochromatin-related proteins, including a nuclear lamina protein, Lamin (Lam), were associated with Wge protein in cultured cells. Knockdown experiments revealed that Lam is indeed required for Wge-mediated eye-to-wing TD. Moreover, Wge over-expression altered the spatial organization of genomic DNA inside the cell nuclei. Accordingly, we suggest that Wge interacts with Lam to link some genomic regions with the nuclear periphery and regulates chromatin dynamics in imaginal disc TD.
RESUMO
Over-expression of Winged-Eye (WGE) in the Drosophila eye imaginal disc induces an eye-to-wing transformation. Endogenous WGE is required for organ development, and wge-deficient mutants exhibit growth arrest at the larval stage, suggesting that WGE is critical for normal growth. The function of WGE, however, remains unclear. Here, we analyzed the subcellular localization of WGE to gain insight into its endogenous function. Immunostaining showed that WGE localized to specific nuclear foci called the histone locus body (HLB), an evolutionarily conserved nuclear body required for S phase-specific histone mRNA production. Histone mRNA levels and protein levels in cytosolic fractions were aberrantly up-regulated in wge mutant larva, suggesting a role for WGE in regulating histone gene expression. Genetic analyses showed that wge suppresses position-effect variegation, and that WGE and a HLB component Mute appears to be synergistically involved in heterochromatin formation. Further supporting a role in chromatin regulation, wge-deficient mutants showed derepression of retrotransposons and increased γH2Av signals, a DNA damage marker. These findings suggest that WGE is a component of HLB in Drosophila with a role in heterochromatin formation and transposon silencing. We propose that WGE at HLB contributes to genomic stability and development by regulating heterochromatin structure via histone gene regulation.
Assuntos
Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Instabilidade Genômica , Histonas/metabolismo , Animais , Elementos de DNA Transponíveis , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Discos Imaginais/metabolismo , Larva/genética , Larva/metabolismo , Mutação , RNA Interferente Pequeno/metabolismoRESUMO
Drosophila imaginal discs are an excellent model system for studies of developmental plasticity. In imaginal discs, most cells adhere strictly to their specific identity, but some cells undergo transdetermination, a process wherein the determined identity switches to another disc-specific identity. In this study, we performed gain-of-function screening and identified a gene, CG17836/Xrp1, that induces ectopic antennae in the eye field upon overexpression at the early eye disc stage. An essential factor in the distalization process, Distalles, and its upstream regulators Wingless, Hedgehog, and Decapentaplegic, are ectopically induced by CG17836/Xrp1 overexpression in eye discs, and this provides molecular evidence of the formation of ectopic antennae. Further, forced expression of CG17836/Xrp1 induced severe cell-proliferation defects. These findings suggest that CG17836/Xrp1 is involved in the regulation of cell proliferation in eye discs and affects disc identity specification.
Assuntos
Antenas de Artrópodes/metabolismo , Olho Composto de Artrópodes/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Discos Imaginais/metabolismo , Animais , Antenas de Artrópodes/crescimento & desenvolvimento , Proliferação de Células , Transdiferenciação Celular , Olho Composto de Artrópodes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Discos Imaginais/crescimento & desenvolvimento , Morfogênese/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Human remodeling and spacing factor (RSF) consists of a heterodimer of Rsf-1 and hSNF2H, a counterpart of Drosophila ISWI. RSF possesses not only chromatin remodeling activity but also chromatin assembly activity in vitro. While no other single factor can execute the same activities as RSF, the biological significance of RSF remained unknown. To investigate the in vivo function of RSF, we generated a mutant allele of Drosophila Rsf-1 (dRsf-1). The dRsf-1 mutant behaved as a dominant suppressor of position effect variegation. In dRsf-1 mutant, the levels of histone H3K9 dimethylation and histone H2A variant H2Av were significantly reduced in an euchromatic region juxtaposed with heterochromatin. Furthermore, using both genetic and biochemical approaches, we demonstrate that dRsf-1 interacts with H2Av and the H2Av-exchanging machinery Tip60 complex. These results suggest that RSF contributes to histone H2Av replacement in the pathway of silent chromatin formation.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Sequência de Bases , Montagem e Desmontagem da Cromatina , Primers do DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Eucromatina/genética , Eucromatina/metabolismo , Inativação Gênica , Genes de Insetos , Histonas/química , Mutação , Domínios e Motivos de Interação entre Proteínas , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Epigenetic mechanisms are thought to help regulate the unique transcription program that is established in germ cell development. During the germline cycle of many organisms, the epigenome undergoes waves of extensive resetting events, while a part of epigenetic modification remains faithful to specific loci. Little is known about the mechanisms underlying these events, how loci are selected for, or avoid, reprogramming, or even why these events are required. In particular, although the significance of genomic imprinting phenomena involving DNA methylation in mammals is now well accepted, the role of histone modification as a transgenerational epigenetic mechanism has been the subject of debate. Such epigenetic mechanisms may help regulate transcription programs and/or the pluripotent status conferred on germ cells, and contribute to germ line continuity across generations. Recent studies provide new evidence for heritability of histone modifications through germ line cells and its potential effects on transcription regulation both in the soma and germ line of subsequent generations. Unraveling transgenerational epigenetic mechanisms involving highly conserved histone modifications in elegant model systems will accelerate the generation of new paradigms and inspire research in a wide variety of fields, including basic developmental studies and clinical stem cell research.
Assuntos
Desenvolvimento Embrionário/fisiologia , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/citologia , Histonas/metabolismo , Padrões de Herança/genética , Modelos Biológicos , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Oxirredutases N-Desmetilantes/metabolismoRESUMO
Drosophila imaginal disc cells exhibit a remarkable ability to convert cell fates in response to various perturbations, a phenomenon called transdetermination (TD). We previously identified winged eye (wge) as a factor that induces eye-to-wing TD upon overexpression in eye imaginal discs, but the molecular mechanisms underlying TD have remained largely unclear. Here, we found that wge induces various histone modifications and enhances the methylation of Lys9 on histone H3 (H3K9), a feature of heterochromatin. A histone methyltransferase, Su(var)3-9, is required for wge-mediated H3K9 methylation and eye-to-wing TD. Su(var)3-9 is also required for classical wound-induced TD but not for normal development, suggesting its involvement in several types of imaginal disc TDs. Transcriptome analysis revealed that wge represses eye identity genes independently of Su(var)3-9 and activates TD-related genes by acting together with Su(var)3-9. These findings provide new insights into diverse types of chromatin regulation at progressive steps of cell-fate conversions.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Discos Imaginais/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , Discos Imaginais/citologia , Metilação , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: The processes through which the germline maintains its continuity across generations has long been the focus of biological research. Recent studies have suggested that germline continuity can involve epigenetic regulation, including regulation of histone modifications. However, it is not clear how histone modifications generated in one generation can influence the transcription program and development of germ cells of the next. RESULTS: We show that the histone H3K36 methyltransferase maternal effect sterile (MES)-4 is an epigenetic modifier that prevents aberrant transcription activity in Caenorhabditis elegans primordial germ cells (PGCs). In mes-4 mutant PGCs, RNA Pol II activation is abnormally regulated and the PGCs degenerate. Genetic and genomewide analyses of MES-4-mediated H3K36 methylation suggest that MES-4 activity can operate independently of ongoing transcription, and may be predominantly responsible for maintenance methylation of H3K36 in germline-expressed loci. CONCLUSIONS: Our data suggest a model in which MES-4 helps to maintain an 'epigenetic memory' of transcription that occurred in germ cells of previous generations, and that MES-4 and its epigenetic product are essential for normal germ cell development.
RESUMO
DNA supercoiling factor (SCF) generates unconstrained negative supercoils of DNA in conjunction with eukaryotic topoisomerase II. In Drosophila melanogaster, SCF localizes to puffs on polytene chromosomes and is required for dosage compensation via hypertranscripton of genes on the male X chromosome. The present study investigated the role of SCF on autosomes. Although RNAi knockdown of scf results in male lethality, some escapers showed anterior homeotic transformation of the male sixth abdominal segment, similar to that arising from reduced expression of Abdominal-B (Abd-B). Heterozygotes for an scf mutant allele (scf(1)) displayed suppression of Pc mutation-dependent posterior transformation and enhancement of anterior transformation caused by trxG mutations. The level of Abd-B mRNA decreased in scf(1) embryos compared with wild-type. Tiling array experiments showed the presence of significant SCF signals in an Abd-B promoter region. Expression from the basal Abd-B promoter on a transgene was reduced in scf(1) embryos compared with wild-type. Collectively, these results demonstrate that SCF occupies the promoter region of Abd-B and activates expression for the proper formation of abdominal segments. Furthermore, preferential occupancy of SCF around transcription start sites of many active genes suggests a role for the factor in positive regulation of promoters.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas Nucleares/metabolismo , Abdome/embriologia , Abdome/inervação , Animais , DNA Super-Helicoidal/metabolismo , Drosophila/embriologia , Drosophila melanogaster/genética , Genes Homeobox , Genes de Insetos , Proteínas de Homeodomínio/metabolismo , Análise em MicrossériesRESUMO
DNA supercoiling factor (SCF) is a protein capable of generating negative supercoils in DNA in conjunction with topoisomerase II. To clarify the biological functions of SCF, we introduced a heritable SCF RNAi into Drosophila. Upon knockdown of SCF, we observed male lethality and male-specific reduction in the expression levels of X-linked genes. SCF functionally interacts with components of the MSL complex, which are required for dosage compensation via hypertranscription of the male X chromosome. Moreover, SCF colocalizes with the MSL complex along the male X chromosome. Upon overexpression of SCF, the male X chromosome had a bloated appearance. This phenotype was dependent on the histone acetyltransferase MOF and was suppressed by simultaneous overexpression of ISWI. These findings demonstrate that SCF plays a role in transcriptional activation via alteration of chromatin structure and provide evidence that SCF contributes to dosage compensation.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Cromatina/metabolismo , Mecanismo Genético de Compensação de Dose/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/genética , Regulação da Expressão Gênica , Genes Ligados ao Cromossomo X/genética , Proteínas Nucleares/fisiologia , Cromossomo X/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Cromatina/genética , Primers do DNA , Mecanismo Genético de Compensação de Dose/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histona Acetiltransferases/metabolismo , Immunoblotting , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Cromossomo X/genéticaRESUMO
The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.