RESUMO
In eukaryotes the TFIID complex is required for preinitiation complex assembly which positions RNA polymerase II around transcription start sites. On the other hand, histone acetyltransferase complexes including SAGA and ATAC, modulate transcription at several steps through modification of specific core histone residues. In this study we investigated the function of Drosophila melanogaster proteins TAF10 and TAF10b, which are subunits of dTFIID and dSAGA, respectively. We generated a mutation which eliminated the production of both Drosophila TAF10 orthologues. The simultaneous deletion of both dTaf10 genes impaired the recruitment of the dTFIID subunit dTAF5 to polytene chromosomes, while binding of other TFIID subunits, dTAF1 and RNAPII was not affected. The lack of both dTAF10 proteins resulted in failures in the larval-pupal transition during metamorphosis and in transcriptional reprogramming at this developmental stage. Surprisingly, unlike dSAGA mutations, dATAC subunit mutations resulted in very similar changes in the steady state mRNA levels of approximately 5000 genes as did ablation of both dTaf10 genes, indicating that dTAF10- and/or dTAF10b-containing complexes and dATAC affect similar pathways. Importantly, the phenotype resulting from dTaf10+dTaf10b mutation could be rescued by ectopically added ecdysone, suggesting that dTAF10- and/or dTAF10b-containing complexes are involved in the expression of ecdysone biosynthetic genes. Indeed, in dTaf10+dTaf10b mutants, cytochrome genes, which regulate ecdysone synthesis in the ring gland, were underrepresented. Therefore our data support the idea that the presence of dTAF10 proteins in dTFIID and/or dSAGA is required only at specific developmental steps. We propose that distinct forms of dTFIID and/or dSAGA exist during Drosophila metamorphosis, wherein different TAF compositions serve to target RNAPII at different developmental stages and tissues.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Ecdisona/metabolismo , Larva/fisiologia , Metamorfose Biológica/fisiologia , Pupa/fisiologia , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Proteínas de Drosophila/genética , Ecdisona/genética , Mutação , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genéticaRESUMO
Multipotent precursors are plastic cells that generate different, stable fates at the correct number, place and time, to allow tissue and organ formation. While fate determinants are known to trigger specific transcriptional programs, the molecular pathway driving the progression from multipotent precursors towards stable and specific identities remains poorly understood. Here we demonstrate that, in Drosophila neural precursors, the glial determinant glial cell missing (Gcm) acts as a 'time bomb' and triggers its own degradation once the glial programme is stably activated. This requires a sequence of transcriptional and posttranscriptional loops, whereby a Gcm target first affects the expression and then acetylation of the fate determinant, thus controlling Gcm levels and stability over time. Defective homeostasis between the loops alters the neuron:glia ratio and freezes cells in an intermediate glial/neuronal phenotype. In sum, we identify an efficient strategy triggering cell identity, a process altered in pathological conditions such as cancer.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Animais , Linhagem da Célula , Drosophila/citologia , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: ADA2 proteins, together with ADA3, SGF29 and GCN5 form the acetyltransferase module of GNAT-type histone acetyltransferase complexes. ADA2b is present in the SAGA complex, which plays roles in various chromatin-related processes via histone H3 modifications and by other mechanisms. RESULTS: In this report we present findings showing that during Drosophila melanogaster development two dADA2b isoforms (dADA2bS and dADA2bL) - which differ in their C-terminal domains - are expressed at various levels. Genetic complementation experiments indicate that dADA2bS alone can support development but cannot fully complement dAda2b mutations. In the presence of dADA2bS, the SAGA-specific histone H3 acetylation level is partially restored in dAda2b mutants. Comparison of whole transcriptome profiles of dAda2b null and dAda2bS transgene-carrier dAda2b null larvae indicates partial overlap between affected genes. mRNA levels corresponding to selected genes which are either up- or down-regulated in dAda2b mutants are altered by dADA2bS expression to different extents, ranging from complete restoration to wild type levels to no restoration at all. The short (dADA2bS) isoform of dADA2b seems to be more capable of restoring lost dSAGA functions that cause mRNA level up-regulation than those that lead to decreased mRNA levels. CONCLUSIONS: The data presented here are in accord with results of genetic complementation experiments, and support the hypothesis that different isoforms of dADA2b contribute to the functional variations of dSAGA multiprotein HAT complexes.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Acetilação , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Transcrição GênicaRESUMO
In Drosophila, the transcription factor Gcm/Glide plays a key role in cell fate determination and cellular differentiation. In light of its crucial biological impact, major efforts have been put for analyzing its properties as master regulator, from both structural and functional points of view. However, the lack of efficient antibodies specific to the Gcm/Glide protein precluded thorough analyses of its regulation and activity in vivo. In order to relieve such restraints, we designed an epitope-tagging approach to "FLAG"-recognize and analyze the functional protein both in vitro (exogenous Gcm/Glide) and in vivo (endogenous protein). We here (i) reveal a tight interconnection between the small RNA and the Gcm/Glide pathways. AGO1 and miR-1 are Gcm/Glide targets whereas miR-279 negatively controls Gcm/Glide expression (ii) identify a novel cell population, peritracheal cells, expressing and requiring Gcm/Glide. Peritracheal cells are non-neuronal neurosecretory cells that are essential in ecdysis. In addition to emphasizing the importance of following the distribution and the activity of endogenous proteins in vivo, this study provides new insights and a novel frame to understand the Gcm/Glide biology.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Argonautas/metabolismo , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Desenvolvimento Embrionário , Proteínas de Fluorescência Verde/genética , Muda , Mutação , Sistema Nervoso/embriologia , Fatores de Transcrição/genéticaRESUMO
Posttranslational modification of histones regulates transcription but the exact role that acetylation of specific lysine residues plays in biological processes in vivo is still not clearly understood. To assess the contribution of different histone modifications to transcriptional activation in vivo, we determined the acetylation patterns on the ecdysone induced Eip74EF and Eip75B genes in Drosophila melanogaster larvae by chromatin immunoprecipitation. We found that acetylation of histone H3 lysine 23 is localized to promoters and correlates with endogenous ecdysone induced gene activation. In contrast, acetylation of lysines 8, 12 and 16 of histone H4 and lysine 9 of histone H3 showed minor differences in their distribution on the regulatory and transcribed regions tested, and had limited or no correlation with ecdysone induced transcriptional activity. We found that dCBP, which is encoded by the nejire gene, acetylates H3 lysine 23 in vivo, and silencing of nejire leads to reduced expression of the Eip74EF and Eip75B genes. Our results suggest that acetylation of specific lysine residues of histones contribute specifically to the dynamic regulation of transcription. Furthermore, along with previous studies identify CBP dependent H3 lysine 23 acetylation as an evolutionarily conserved chromatin modification involved in steroid induced gene activation.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Ecdisona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Lisina/metabolismo , Acetilação/efeitos dos fármacos , Acetiltransferases/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Genes de Insetos/genética , Larva/genética , Larva/crescimento & desenvolvimento , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Recent data from clinical and mammalian genetic studies indicate that COL4A1 mutations manifest with basement membrane defects that result in muscle weakness, cramps, contractures, dystrophy and atrophy. In-depth studies of mutant COL4A1-associated muscle phenotype, however, are lacking and significant details of the muscle-specific pathomechanisms remain unknown. In this study, we have used a comprehensive set of Drosophila col4a1 and col4a2 mutants and a series of genetic and mutational analyses, gene, protein expression, and immunohistochemistry experiments in order to establish a Drosophila model and address some of these questions. The Drosophila genome contains two type IV collagen genes, col4a1 and col4a2. Mutant heterozygotes of either gene are viable and fertile, whereas homozygotes are lethal. In complementation analysis of all known mutants of the locus and a complementation matrix derived from these data we have identified the dominant lesions within the col4a1, but not within the col4a2 gene. Expression of a col4a1 transgene partially rescued the dominant and recessive mutant col4a1 alleles but not the col4a2 mutations that were all recessive. Partial complementation suggested that col4a1 gene mutations have strong antimorph effect likely due to the incorporation of the mutant protein into the triple helix. In col4a1 mutants, morphological changes of the oviduct muscle included severe myopathy with centronuclear myofibers leading to gradual development of female sterility. In larval body wall muscles ultrastructural changes included disturbance of A and I bands between persisting Z bands. In the most severely affected DTS-L3 mutant, we have identified four missense mutations within the coding region of the col4a1 gene two of which affected the Y within the Gly-X-Y unit and a 3' UTR point mutation. In conclusion, our Drosophila mutant series may serve as an effective model to uncover the mechanisms by which COL4A1 mutations result in compromised myofiber-basement membrane interactions and aberrant muscle function.
Assuntos
Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Doenças Musculares/genética , Proteínas Mutantes/genética , Mutação/genética , Animais , Membrana Basal/metabolismo , Modelos Animais de Doenças , Drosophila , Feminino , Heterozigoto , Homozigoto , Doenças Musculares/patologia , Proteínas Mutantes/metabolismo , FenótipoRESUMO
Neurons and glia differentiate from multipotent precursors called neural stem cells (NSCs), upon the activation of specific transcription factors. In vitro, it has been shown that NSCs display very plastic features; however, one of the major challenges is to understand the bases of lineage restriction and NSC plasticity in vivo, at the cellular level. We show here that overexpression of the Gcm transcription factor, which controls the glial versus neuronal fate choice, fully and efficiently converts Drosophila NSCs towards the glial fate via an intermediate state. Gcm acts in a dose-dependent and autonomous manner by concomitantly repressing the endogenous program and inducing the glial program in the NSC. Most NSCs divide several times to build the embryonic nervous system and eventually enter quiescence: strikingly, the gliogenic potential of Gcm decreases with time and quiescent NSCs are resistant to fate conversion. Together with the fact that Gcm is able to convert mutant NSCs that cannot divide, this indicates that plasticity depends on temporal cues rather than on the mitotic potential. Finally, NSC plasticity involves specific chromatin modifications. The endogenous glial cells, as well as those induced by Gcm overexpression display low levels of histone 3 lysine 9 acetylation (H3K9ac) and Drosophila CREB-binding protein (dCBP) Histone Acetyl-Transferase (HAT). Moreover, we show that dCBP targets the H3K9 residue and that high levels of dCBP HAT disrupt gliogenesis. Thus, glial differentiation needs low levels of histone acetylation, a feature shared by vertebrate glia, calling for an epigenetic pathway conserved in evolution.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Histonas/química , Células-Tronco Neurais/citologia , Neuroglia/citologia , Fatores de Transcrição/fisiologia , Acetilação , Animais , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Epigênese Genética , Histonas/metabolismo , Imuno-Histoquímica/métodos , Hibridização In Situ , Mitose , Fatores de Transcrição/metabolismo , Vertebrados/metabolismoRESUMO
In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently.
Assuntos
Drosophila melanogaster/genética , Histonas/metabolismo , Lisina/metabolismo , Acetilação , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Histonas/química , MutaçãoRESUMO
The reversible phosphorylation-dephosphorylation of RNA polymerase II (Pol II) large subunit carboxyl terminal domain (CTD) during transcription cycles in eukaryotic cells generates signals for the steps of RNA synthesis and maturation. The major phosphatase specific for CTD dephosphorylation from yeast to mammals is the TFIIF-interacting CTD-phosphatase, Fcp1. We report here on the in vivo analysis of Fcp1 function in Drosophila using transgenic lines in which the phosphatase production is misregulated. Fcp1 function is essential throughout Drosophila development and ectopic up- or downregulation of fcp1 results in lethality. The fly Fcp1 binds to specific regions of the polytene chromosomes at many sites colocalized with Pol II. In accord with the strong evolutional conservation of Fcp1: (1) the Xenopus fcp1 can substitute the fly fcp1 function, (2) similarly to its S. pombe homologue, Drosophila melanogaster (Dm)Fcp1 interacts with the RPB4 subunit of Pol II, and (3) transient expression of DmFcp1 has a negative effect on transcription in mammalian cells. The in vivo experimental system described here suggests that fly Fcp1 is associated with the transcription engaged Pol II and offers versatile possibilities for studying this evolutionary conserved essential enzyme.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase II/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Linhagem Celular , Cromatina/metabolismo , Clonagem Molecular , Primers do DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Genes de Insetos , Genes Reporter , Células HeLa , Humanos , Masculino , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Polimerase II/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Although telomere formation occurs through a different mechanism in Drosophila compared with other organisms, telomere associations result from mutations in homologous genes, indicating the involvement of similar pathways in chromosome end protection. We report here that mutations of the Drosophila melanogaster gene CG31241 lead to high frequency chromosome end fusions. CG31241 is a bicistronic gene that encodes trimethylguanosine synthase (TGS1), which forms the m3G caps of noncoding small RNAs, and a novel protein, DTL. We show that although TGS1 has no role in telomere protection, DTL is localized at specific sites, including the ends of polytene chromosomes, and its loss results in telomere associations. Mutations of ATM- and Rad3-related (ATR) kinase suppress telomere fusions in the absence of DTL. Thus, genetic interactions place DTL in an ATR-related pathway in telomere protection. In contrast to ATR kinase, mutations of ATM (ataxia telangiectasia mutated) kinase, which acts in a partially overlapping pathway of telomere protection, do not suppress formation of telomere associations in the absence of DTL. Thus, uncovering the role of DTL will help to dissect the evolutionary conserved pathway(s) controlling ATM-ATR-related telomere protection.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Genes de Insetos/genética , Metiltransferases/genética , Telômero/metabolismo , Alelos , Animais , Cromossomos/metabolismo , Drosophila melanogaster/citologia , Neurônios/citologia , Neurônios/enzimologia , Fases de Leitura Aberta/genética , Ligação ProteicaRESUMO
Various combinations of post-translational modifications of the N-terminal tails of nucleosomal histones serve as signals to govern chromatin-related processes. The relationship, however, among different types of histone modifications - most frequently acetylation, phosphorylation and methylation - and the order of their establishment has been explored only in a few cases. Here we show that a reduced level of histone H4 acetylated at Lys12 by the ATAC-HAT complex leads to a decrease in the histone H3 phosphorylation at Ser10 by the kinase JIL-1. As JIL-1 activity antagonizes histone H3 dimethylation at Lys9 by SU(VAR)3-9, our observations demonstrate the interdependent actions of an acetyltransferase, a kinase and a methyltransferase. We demonstrate that, in accord with the steps of modifications, mutations that affect ATAC subunits (such as dGcn5, dAda2a and dAda3) (1) decrease the level histone H3 phosphorylation at Ser10, (2) can be rescued partially by JIL-1 overproduction, (3) enhance the spread of histone H3 dimethylation at Lys9 and (4) are suppressed by mutations of Su(var)3-9. We propose that a reduced level of histone H4 acetylated at Lys12 by ATAC attenuates histone H3 phosphorylation at Ser10 by JIL-1 owing to reduced binding of JIL-1 to hypoacetylated chromatin.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Acetilação , Animais , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , Complexos Multiproteicos/genética , Mutação , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Drosophila Gcn5 is the catalytic subunit of the SAGA and ATAC histone acetylase complexes. Here, we show that mutations in Gcn5 and the ATAC component Ada2a induce a decondensation of the male X chromosome, similar to that induced by mutations in the Iswi and Nurf301 subunits of the NURF nucleosome remodelling complex. Genetic studies as well as transcript profiling analysis indicate that ATAC and NURF regulate overlapping sets of target genes during development. In addition, we find that Ada2a chromosome binding and histone H4-Lys12 acetylation are compromised in Iswi and Nurf301 mutants. Our results strongly suggest that NURF is required for ATAC to access the chromatin and to regulate global chromosome organization.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Histona Acetiltransferases/metabolismo , Complexos Multiproteicos/metabolismo , Cromossomo X/metabolismo , Acetilação , Adenosina Trifosfatases/metabolismo , Animais , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Genes de Insetos , Larva , Masculino , Mutação/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Daxx-like protein (DLP), the Drosophila homolog of Daxx, binds Drosophila melanogaster p53 (Dmp53) through its C-terminal region. We generated DLP mutants and found that although DLP expression is developmentally regulated, it is not essential for the execution of the developmental program. The effects DLP mutations show in the loss of heterozygosity assay and on phenotypes resulting from Dmp53 overexpression indicate a genetic interaction between DLP and Dmp53. In contrast to Dmp53 mutants, however, loss of DLP does not result in radiosensitivity indicating that it does not play an essential role in the activation of Dmp53-dependent response after ionizing radiation, and DLP is also not required for the irradiation-induced activation of reaper. In contrast, DLP is involved in the transcriptional regulation of Ark, because Ark mRNA level is decreased in DLP mutants and increased upon ectopic overexpression of DLP. Interestingly, DLP mutants have reduced longevity and reduced female fertility. Altogether, our data suggest complex functions for DLP, which include an anti-apoptotic effect exerted through repression of some Dmp53 functions, and activation of some proapoptotic genes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Longevidade/fisiologia , Proteínas Nucleares/metabolismo , RNA Mensageiro/biossíntese , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/fisiologia , Apoptose/efeitos da radiação , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Fertilidade/genética , Fertilidade/efeitos da radiação , Proteínas Inibidoras de Apoptose/genética , Longevidade/efeitos da radiação , Perda de Heterozigosidade/genética , Proteínas Nucleares/genética , Fenótipo , RNA Mensageiro/genética , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Transcrição Gênica/efeitos da radiação , Proteína Supressora de Tumor p53/genéticaRESUMO
The histone acetyltransferase (HAT) Gcn5 plays a role in chromatin structure and gene expression regulation as a catalytic component of multiprotein complexes, some of which also contain Ada2-type transcriptional coactivators. Data obtained mostly from studies on yeast (Saccharomyces cerevisiae) suggest that Ada2 potentiates Gcn5 activity and substrate recognition. dAda2b, one of two related Ada2 proteins of Drosophila melanogaster, was recently found to play a role in complexes acetylating histone 3 (H3). Evidence of an in vivo functional link between the related coactivator dAda2a and dGcn5, however, is lacking. Here we present data on the genetic interaction of dGcn5 and dAda2a. The loss of either dGcn5 or dAda2a function results in similar chromosome structural and developmental defects. In dAda2a mutants, the nucleosomal H4 acetylation at lysines 12 and 5 is significantly reduced, while the acetylation established by dAda2b-containing Gcn5 complexes at H3 lysines 9 and 14 is unaffected. The data presented here, together with our earlier data on the function of dAda2b, provide evidence that related Ada2 proteins of Drosophila, together with Gcn5 HAT, are involved in the acetylation of specific lysine residues in the N-terminal tails of nucleosomal H3 and H4. Our data suggest dAda2a involvement in both uniformly distributed H4 acetylation and gene-specific transcription regulation.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Histona Acetiltransferases/fisiologia , Histonas/metabolismo , Nucleossomos/enzimologia , Transcrição Gênica , Acetilação , Animais , Regulação para Baixo/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histona Acetiltransferases/deficiência , Histona Acetiltransferases/genética , Histonas/antagonistas & inibidores , Deleção de Sequência , Especificidade por Substrato/genéticaRESUMO
In Drosophila and several other metazoan organisms, there are two genes that encode related but distinct homologs of ADA2-type transcriptional adaptors. Here we describe mutations of the two Ada2 genes of Drosophila melanogaster. By using mutant Drosophila lines, which allow the functional study of individual ADA2s, we demonstrate that both Drosophila Ada2 genes are essential. Ada2a and Ada2b null homozygotes are late-larva and late-pupa lethal, respectively. Double mutants have a phenotype identical to that of the Ada2a mutant. The overproduction of ADA2a protein from transgenes cannot rescue the defects resulting from the loss of Ada2b, nor does complementation work vice versa, indicating that the two Ada2 genes of Drosophila have different functions. An analysis of germ line mosaics generated by pole-cell transplantation revealed that the Ada2a function (similar to that reported for Ada2b) is required in the female germ line. A loss of the function of either of the Ada2 genes interferes with cell proliferation. Interestingly, the Ada2b null mutation reduces histone H3 K14 and H3 K9 acetylation and changes TAF10 localization, while the Ada2a null mutation does not. Moreover, the two ADA2s are differently required for the expression of the rosy gene, involved in eye pigment production, and for Dmp53-mediated apoptosis. The data presented here demonstrate that the two genes encoding homologous transcriptional adaptor ADA2 proteins in Drosophila are both essential but are functionally distinct.
Assuntos
Acetiltransferases/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Acetilação , Acetiltransferases/genética , Animais , Cromossomos/química , Cromossomos/metabolismo , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Olho/metabolismo , Feminino , Genes de Insetos , Histona Acetiltransferases , Histonas/metabolismo , Mutação , Nucleossomos/metabolismo , Óvulo/metabolismo , Fenótipo , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Fatores Associados à Proteína de Ligação a TATA/análise , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Transativadores/metabolismo , Fator de Transcrição TFIID/análise , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica , Transgenes , Proteína Supressora de Tumor p53RESUMO
PIMT, a transcriptional coactivator which interacts with and enhances nuclear receptor coactivator PRIP function, was identified recently in mammalian cells and suggested to function as a link between two major multiprotein complexes anchored by CBP/p300 and PBP. Here we describe that the gene of the Drosophila homologue of PIMT, designated as Dtl, is closely associated and has an overlapping promoter with a gene encoding another transcriptional coactivator, ADA2a, which in turn participates in GCN5 HAT-containing complexes. Ada2a also produces an RNA polII subunit, RPB4, via alternative splicing; consequently, an overlapping regulatory region serves for the production of three proteins, each involved in transcription. By studying expression of reporter gene fusions in tissue culture cells and transgenic animals we have demonstrated that the regulatory regions of Ada2a/Rpb4 and Dtl overlap and the Dtl promoter is partly within the Ada2a/Rpb4 coding region. The shared regulatory region contains a DRE element, binding site of DREF, the protein factor involved in the regulation of a number of genes which play a role in DNA replication and cell proliferation. Despite the perfectly symmetrical DRE, DREF seems to have a more decisive role in Ada2a/Rpb4 transcription than in the transcription of Dtl.
Assuntos
Acetiltransferases/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Drosophila/citologia , Drosophila/embriologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ordem dos Genes , Ligação Genética , Histona Acetiltransferases , Óperon Lac/genética , Luciferases/genética , Luciferases/metabolismo , Masculino , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transfecção , beta-Galactosidase/metabolismoRESUMO
We describe a novel Drosophila gene, dtl (Drosophila Tat-like), which encodes a 60-kDa protein with RNA binding activity and a methyltransferase (MTase) domain. Dtl has an essential role in Drosophila development. The homologs of DTL recently described include PIMT (peroxisome proliferator-activated receptor-interacting protein with a methyltransferase domain), an RNA-binding protein that interacts with and enhances the nuclear receptor coactivator function, and TGS1, the methyltransferase involved in the formation of the 2,2,7-trimethylguanosine (m3G) cap of non-coding small RNAs. DTL is expressed throughout all of the developmental stages of Drosophila. The dtl mRNA has two ORFs (uORF and dORF). The product of dORF is the 60-kDa PIMT/TGS1 homolog protein that is translated from an internal AUG located 538 bp downstream from the 5' end of the message. This product of dtl is responsible for the formation of the m3G cap of small RNAs of Drosophila. Trimethylguanosine synthase activity is essential in Drosophila. The deletion in the dORF or point mutation in the putative MTase active site results in a reduced pool of m3G cap-containing RNAs and lethality in the early pupa stage. The 5' region of the dtl message also has the coding capacity (uORF) for a 178 amino acid protein. For complete rescue of the lethal phenotype of dtl mutants, the presence of the entire dtl transcription unit is required. Transgenes that carry mutations within the uORF restore the MTase activity but result in only partial rescue of the lethal phenotype. Interestingly, two transgenes bearing a mutation in uORF or dORF in trans can result in complete rescue.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/química , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Northern Blotting , Western Blotting , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Proteínas de Drosophila/química , Escherichia coli/metabolismo , Deleção de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Metiltransferases/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Mutação , Fases de Leitura Aberta , Fenótipo , Plasmídeos/metabolismo , Mutação Puntual , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/química , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.