RESUMEN
PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways.
Asunto(s)
ADP-Ribosilación , Interferones , Poli(ADP-Ribosa) Polimerasas , Ubiquitina-Proteína Ligasas , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Interferones/metabolismo , Ubiquitinación , Células HEK293 , SARS-CoV-2/metabolismo , Transducción de Señal , COVID-19/virología , COVID-19/metabolismo , Proteínas de NeoplasiasRESUMEN
PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.
Asunto(s)
COVID-19 , Transferasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Antivirales , Hidrolasas , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.
Asunto(s)
Drosophila , Serina , Animales , Serina/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , ADP-Ribosilación , Poli Adenosina Difosfato Ribosa/química , Adenosina Difosfato Ribosa/metabolismo , Mamíferos/metabolismoRESUMEN
In human cells, appropriate monomethylation of histone H4 lysine 20 by PrSet7 (also known as SET8 and SETD7) is important for the correct transcription of specific genes and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with proliferating cell nuclear antigen (PCNA), which targets PrSet7 for destruction through the pathway mediated by CRL4(C) (dt2) (the cullin ring finger ligase-4 complex containing Cdt2). However, the factors involved in positive regulation of PrSet7 histone methylation remain undefined. Here, we present biochemical and genetic evidence for a previously undocumented interaction between Drosophila PrSet7 (dPrSet7) and DNA polymerase α in Drosophila. Depletion of the polymerase reduces H4K20 monomethylation suggesting that it is required for dPrSet7 histone methylation activity. We also show that the interaction between PCNA and PrSet7 is conserved in Drosophila, but is only detectable in chromatin fractions. Consistent with this, S2 cells show a significant loss of chromatin-bound dPrSet7 protein as S phase progresses. Based on these data we suggest that interaction with the DNA polymerase represents an important route for stimulation of PrSet7 histone methylase activity that is mediated by allowing loading of dPrSet7 onto chromatin or its subsequent activation.
Asunto(s)
ADN Polimerasa I/metabolismo , Drosophila/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Animales , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , ADN Polimerasa I/química , ADN Polimerasa I/genética , Drosophila/química , Drosophila/genética , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Histonas/genéticaRESUMEN
DNA polymerase ε (polε) plays a central role in DNA replication in eukaryotic cells, and has been suggested to the main synthetic polymerase on the leading strand. It is a hetero-tetrameric enzyme, comprising a large catalytic subunit (the A subunit ~250 kDa), a B subunit of ~60 kDa in most species (~80 kDa in budding yeast) and two smaller subunits (each ~20 kDa). In Drosophila, two subunits of polε (dpolε) have been identified. One is the 255 kDa catalytic subunit (dpolεp255), and the other is the 58 kDa subunit (dpolεp58). The functions of the B subunit have been mainly studied in budding yeast and mammalian cell culture, few studies have been performed in the context of an intact multicellular organism and therefore its functions in this context remain poorly understood. To address this we examined the in vivo role of dpolεp58 in Drosophila. A homozygous dpolεp58 mutant is pupal lethal, and the imaginal discs are less developed in the third instar larvae. In the eye discs of this mutant S phases, as measured by BrdU incorporation assays, were significantly reduced. In addition staining with an anti-phospho histone H3 (PH3) antibody, (a marker of M phase), was increased in the posterior region of eye discs, where usually cells stop replicating and start differentiation. These results indicate that dpolεp58 is essential for Drosophila development and plays an important role in progression of S phase in mitotic cell cycles. We also observed that the size of nuclei in salivary gland cells were decreased in dpolεp58 mutant, indicating that dpolεp58 also plays a role in endoreplication. Furthermore we detect a putative functional interaction between dpolε and ORC2 in discs suggesting that polε plays a role in the initiation of DNA replication in Drosophila.
RESUMEN
Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in microorganisms, insects, plants, and mammals. The mammalian enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The present study reports that eye imaginal disc-specific knockdown of the Drosophila HO homologue (dHO) conferred serious abnormal eye morphology in adults, resulting in the generation of reactive oxygen species and apoptosis in third-instar larvae. Oxidative stress frequently induces DNA lesions that are recognized by damage sensors, including ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and rad3-related (ATR) proteins. The knockdown of dHO took place in G0/G1-arrested cells posterior to the morphogenetic furrow and thus prevented these cells from entering S-phase, with an increase in the level of histone H2A.V, a DNA damage marker. Moreover, the knockdown of dHO resulted in the enhancement of the rough eye phenotype in ATM-deficient flies or was lethal in ATR-deficient flies. These results indicate that dHO functions in control of the signal pathway of DNA damage. On the other hand, genetic crosses with a collection of Drosophila deficiency stocks allowed us to identify eight genomic regions, each deletion of which caused suppression of the rough eye phenotype induced by dHO knockdown. This information should facilitate the identification of HO regulators in Drosophila and clarification of the roles of HO in eye development.
Asunto(s)
Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Daño del ADN/genética , Drosophila melanogaster/enzimología , Hemo Oxigenasa (Desciclizante)/metabolismo , Transducción de Señal/genética , Animales , Animales Modificados Genéticamente , Bromodesoxiuridina , Ciclo Celular/genética , Ciclo Celular/fisiología , Ojo Compuesto de los Artrópodos/anomalías , Cruzamientos Genéticos , Daño del ADN/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Técnicas de Silenciamiento del Gen , Células HEK293 , Hemo Oxigenasa (Desciclizante)/genética , Humanos , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Discos Imaginales/ultraestructura , Larva/enzimología , Larva/crecimiento & desarrollo , Microscopía Electrónica de Rastreo , Interferencia de ARN , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno , Transducción de Señal/fisiologíaRESUMEN
Nuclear transcription factor Y (NF-Y) is well characterized in eukaryotes. It consists of three different subunits, NF-YA, NF-YB and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. There is a high homology in NF-YB among Drosophila species with 75% identity and 95% similarity overall, especially in the histone-fold motif (HFM) (95% identity and 100% similarity). In the present study, specific knockdown of Drosophila NF-YB (dNF-YB) in eye imaginal discs induced a rough eye phenotype in adults and this phenotype was the result of induction of caspase-dependent apoptosis followed by apoptosis-induced proliferation. Furthermore, knockdown specifically inhibited R7 photoreceptor cell differentiation, independent of the apoptotic function. dNF-YB and dNF-YA indeed form complexes in vivo where they impair R7 photoreceptor cell differentiation by down regulating the mitogen-activated protein kinase (MAPK) pathway. Expression of the sev gene, or the D-raf gene, a downstream component of the MAPK cascade, could rescue the rough eye phenotype and the loss of R7 signals in dNF-YB knockdown flies. The death executioner Bcl-2 (debcl) is the homolog of Bcl-2 in Drosophila melanogaster and its promoter contains four dNF-Y-binding consensus sequences which play positive roles in promoter activity. In chromatin immunoprecipitation assays with anti-dNF-YB antibody and S2 cells, the debcl gene promoter region containing the NF-Y consensus was effectively amplified in immunoprecipitates by polymerase chain reaction. Taken together, these results indicate that dNF-Y regulates debcl gene expression.
Asunto(s)
Factor de Unión a CCAAT/fisiología , Diferenciación Celular/genética , Drosophila melanogaster/embriología , Ojo/embriología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Factor de Unión a CCAAT/genética , Muerte Celular/genética , Línea Celular , Drosophila melanogaster/genética , Embrión no Mamífero , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-bcl-2/genética , Homología de Secuencia , TransfecciónRESUMEN
In Drosophila, the 255kDa catalytic subunit (dpolεp255) and the 58kDa subunit of DNA polymerase ε (dpolεp58) have been identified. The N-terminus of dpolεp255 carries well-conserved six DNA polymerase subdomains and five 3'â5' exonuclease motifs as observed with Polε in other species. We here examined roles of dpolεp255 during Drosophila development using transgenic fly lines expressing double stranded RNA (dsRNA). Expression of dpolεp255 dsRNA in eye discs induced a small eye phenotype and inhibited DNA synthesis, indicating a role in the G1-S transition and/or S-phase progression of the mitotic cycle. Similarly, expression of dpolεp255 dsRNA in the salivary glands resulted in small size and endoreplication defects, demonstrating a critical role in endocycle progression. In the eye disc, defects induced by knockdown of dpolεp255 were rescued by overexpression of the C-terminal region of dpolεp255, indicating that the function of this non-catalytic domain is conserved between yeast and Drosophila. However, this was not the case for the salivary gland, suggesting that the catalytic N-terminal region is crucial for endoreplication and its defect cannot be complemented by other DNA polymerases. In addition, several genetic interactants with dpolεp255 including genes related to DNA replication such as RFC, DNA primase, DNA polη, Mcm10 and Psf2 and chromatin remodeling such as Iswi were also identified.
Asunto(s)
Ciclo Celular/genética , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , Drosophila/fisiología , Animales , Animales Modificados Genéticamente , Dominio Catalítico/genética , Proteínas de Ciclo Celular/genética , Replicación del ADN , Drosophila/citología , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Larva , Proteínas de Mantenimiento de Minicromosoma , Subunidades de Proteína , ARN Bicatenario , Fase S/genética , Glándulas Salivales/enzimologíaRESUMEN
A novel substrate of Akt/PKB designated as Girdin (griders of actin filaments) has been identified in mammals and characterized as an actin-binding protein. A Girdin-like protein has been identified in Drosophila, which has two isoforms, dGirdin PA and dGirdin PB. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size and this was enhanced by half reduction of the Akt gene dose. Furthermore the dGirdin-knockdown wing disc cells exhibited severe disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway. Human Girdin plays important roles in cancer progression and angiogenesis. Therefore Girdin and its interacting proteins could be potential pharmaceutical targets for cancer therapies and tumor angiogenesis. Possible use of the Drosophila Girdin model in understanding the mechanisms of cancer progression and in developing preventive and therapeutic strategies will be discussed.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/genética , Neoplasias/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/citología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de Microfilamentos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Transporte Vesicular , Citoesqueleto de Actina , Animales , Apoptosis , Tamaño de la Célula , Humanos , Organismos Modificados Genéticamente , Homología de Secuencia de Aminoácido , Alas de Animales/citologíaRESUMEN
The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-beta-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Proteínas de Drosophila/genética , Regiones Promotoras Genéticas , Elementos Reguladores de la Transcripción , Factores de Transcripción/genética , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Drosophila , Fosfatasa 1 de Especificidad Dual/genética , Datos de Secuencia Molecular , Transducción de Señal , Alas de Animales/crecimiento & desarrolloRESUMEN
The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. By genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express DREF in the eye discs, we identified 24 genes that suppressed and 12 genes that enhanced the rough eye phenotype when heterozygous for mutations. Five genes, HP6, pigeon, lace, X box binding protein 1 and guftagu were found to carry replication-related element (DRE) sequences in their 5'-flanking regions. Of these, the HP6 gene carries two sequences that match seven out of eight nucleotides of DRE and two additional sequences that match six out of eight nucleotides of DRE in the 5'-flanking region. Band mobility shift assays using Drosophila Kc cell nuclear extracts demonstrated DREF binding to two of these sites and chromatin immunoprecipitation using anti-DREF antibodies confirmed that this occurs in vivo. Knockdown of DREF in Drosophila S2 cells decreased the HP6 mRNA level. The results, taken together, indicate that DREF directly regulates expression of the HP6 gene. HP6 mRNA was detected throughout development by RT-PCR with highest levels in adult males. In addition, immunostaining analyses revealed colocalization of HP6 and DREF in nuclei at the apical tips in the testes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genes de Insecto , Factores de Transcripción/metabolismo , Animales , Cromosomas/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestructura , Ojo/ultraestructura , Genes Supresores , Masculino , Mutación , Fenotipo , Interferencia de ARN , ARN Mensajero/metabolismo , Testículo/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in micro-organisms, insects, plants, and mammals. The mammalian enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The present study reports the use of RNA-interference (RNAi) to suppress HO in the multicellular eukaryote Drosophila. Eye imaginal disc-specific suppression of the Drosophila HO homolog (dHO) conferred serious abnormal eye morphology in adults. Deficiency of the dHO protein resulted in increased levels of iron and heme in larvae. The accumulation of iron was also observed in the compound eyes of dHO-knockdown adult flies. In parallel with the decrease of dHO, the expression of delta-aminolevulinic acid synthase, the first enzyme of the heme-biosynthetic pathway, in larvae was decreased markedly, suggesting that heme biosynthesis was totally suppressed by dHO-deficiency. The activation of caspase-3 occurred in eye imaginal discs of dHO-knockdown flies, indicating the occurrence of apoptosis in the discs. On the other hand, the overexpression of dHO resulted in a weak but significant rough eye phenotype in adults. Taken together, considering that dHO is not a stress-inducible protein, the expression of dHO can be tightly regulated at developmental stages and the relevant expression is necessary for the normal development of tissues in Drosophila.
Asunto(s)
Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/crecimiento & desarrollo , Hemo Oxigenasa (Desciclizante)/biosíntesis , Animales , Arsenitos/farmacología , Cadmio/farmacología , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/enzimología , Ojo/efectos de los fármacos , Ojo/enzimología , Ojo/crecimiento & desarrollo , Expresión Génica , Técnicas de Silenciamiento del Gen , Hemo Oxigenasa (Desciclizante)/genética , Hemina/farmacologíaRESUMEN
The CCAAT motif-binding factor, nuclear factor Y (NF-Y) consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in the notum compartment of wing discs by a pannir-GAL4 and UAS-dNF-YAIR mainly resulted in a thorax disclosed phenotype. Reduction of the Drosophila c-Jun N-terminal kinase (JNK) basket (bsk) gene dose enhanced the knockdown of dNF-YA-induced phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed reduction in the level of the JNK reporter, puc-LacZ signals, in dNF-YA RNAi clones. In addition, expression of wild-type Bsk effectively suppressed the phenotype induced by knockdown of dNF-YA. The bsk gene promoter contains a CCAAT motif and this motif plays a positive role in the promoter activity. We performed chromatin immunoprecipitation (ChIP) assays in S2 cells with anti-dNF-YA IgG and quantitative real-time PCR. The bsk gene promoter region containing the CCAAT boxes was effectively amplified in the immunoprecipitates by PCR. However, this region was not amplified in the immunoprecipitates from dNF-YA knockdown cells. Furthermore, the level of endogenous bsk mRNA is reduced in the dNF-YA knockdown larvae. These results suggest that dNF-Y is necessary for proper bsk expression and activity of JNK pathway during thorax development.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Factor de Unión a CCAAT/antagonistas & inhibidores , Factor de Unión a CCAAT/genética , Cartilla de ADN/genética , Drosophila/genética , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Genes de Insecto , Plásmidos/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Tórax/crecimiento & desarrollo , Alas de Animales/crecimiento & desarrolloRESUMEN
The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB, and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. NF-YA contains a DNA binding domain in its C-terminal region. We established transgenic fly lines carrying the UAS-HA-dNF-YA or UAS-dNF-YAIR and showed over-expression or knockdown with various GAL4 drivers to be lethal at various developmental stages, suggesting that dNF-YA participate in various gene regulatory pathways during Drosophila development. Expression of dNF-YA with eyeless-GAL4 mainly resulted in lethality with a headless phenotype in pharate-adults. Reduction of the eyeless gene dose enhanced the dNF-YA-induced phenotype, while reduction of the Distal-less gene dose suppressed the phenotype. On the other hand, crossing the dNF-YA over-expressing flies with Notch mutant resulted in no apparent effect on the phenotype. These results suggest that dNF-YA can disturb eye disc specification, but not eye disc growth.