RESUMO
We define a quantitative relationship between the affinity with which the intestine-specific GATA factor ELT-2 binds to cis-acting regulatory motifs and the resulting transcription of asp-1, a target gene representative of genes involved in Caenorhabditis elegans intestine differentiation. By establishing an experimental system that allows unknown parameters (e.g. the influence of chromatin) to effectively cancel out, we show that levels of asp-1 transcripts increase monotonically with increasing binding affinity of ELT-2 to variant promoter TGATAA sites. The shape of the response curve reveals that the product of the unbound ELT-2 concentration in vivo [i.e. (ELT-2free) or ELT-2 'activity'] and the largest ELT-XXTGATAAXX association constant (Kmax) lies between five and ten. We suggest that this (unitless) product [Kmax×(ELT-2free) or the equivalent product for any other transcription factor] provides an important quantitative descriptor of transcription-factor/regulatory-motif interaction in development, evolution and genetic disease. A more complicated model than simple binding affinity is necessary to explain the fact that ELT-2 appears to discriminate in vivo against equal-affinity binding sites that contain AGATAA instead of TGATAA.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fatores de Transcrição GATA/metabolismo , Mucosa Intestinal/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Sequência de Bases , Sítios de Ligação , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Ensaio de Desvio de Mobilidade Eletroforética , Cinética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Ligação Proteica , Transcrição GênicaRESUMO
The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of â¼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen's design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10-4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Diferenciação Celular , Endoderma/metabolismo , Fatores de Transcrição GATA/genética , Genes Modificadores , Intestinos/citologia , Mutação/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular/genética , Embrião não Mamífero/metabolismo , Fatores de Transcrição GATA/química , Fatores de Transcrição GATA/metabolismo , Testes Genéticos , Genótipo , Reprodutibilidade dos Testes , Análise de Sobrevida , Sequenciamento Completo do Genoma , Zigoto/metabolismoRESUMO
The two GATA transcription factors ELT-2 and ELT-7 function in the differentiation of the C. elegans intestine. ELT-2 loss causes lethality. ELT-7 loss causes no obvious phenotype but enhances the elt-2(-) intestinal phenotype. Thus, ELT-2 and ELT-7 appear partially redundant, with ELT-2 being more influential. To investigate the different regulatory roles of ELT-2 and ELT-7, we compared the transcriptional profiles of pure populations of wild-type, elt-2(-), elt-7(-), and elt-7(-); elt-2(-) double mutant L1-stage larvae. Consistent with the mutant phenotypes, loss of ELT-2 had a>25 fold greater influence on the number of significantly altered transcripts compared to the loss of ELT-7; nonetheless, the levels of numerous transcripts changed upon loss of ELT-7 in the elt-2(-) background. The quantitative responses of individual genes revealed a more complicated behaviour than simple redundancy/partial redundancy. In particular, genes expressed only in the intestine showed three distinguishable classes of response in the different mutant backgrounds. One class of genes responded as if ELT-2 is the major transcriptional activator and ELT-7 provides variable compensatory input. For a second class, transcript levels increased upon loss of ELT-2 but decreased upon further loss of ELT-7, suggesting that ELT-7 actually overcompensates for the loss of ELT-2. For a third class, transcript levels also increased upon loss of ELT-2 but remained elevated upon further loss of ELT-7, suggesting overcompensation by some other intestinal transcription factor(s). In spite of its minor loss-of-function phenotype and its limited sequence similarity to ELT-2, ELT-7 expressed under control of the elt-2 promoter is able to rescue elt-2(-) lethality. Indeed, appropriately expressed ELT-7, like appropriately expressed ELT-2, is able to replace all other core GATA factors in the C. elegans endodermal pathway. Overall, this study focuses attention on the quantitative intricacies behind apparent redundancy or partial redundancy of two related transcription factors.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Endoderma/metabolismo , Fatores de Transcrição GATA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Fatores de Transcrição GATA/deficiência , Fatores de Transcrição GATA/genética , Genes de Helmintos , Genes Reporter , Estudos de Associação Genética , Intestinos/citologia , Larva , Regiões Promotoras Genéticas , Transcrição Gênica , TranscriptomaRESUMO
The ELT-2 GATA factor is the predominant transcription factor regulating gene expression in the C. elegans intestine, following endoderm specification. We comment on our previous study (Wiesenfahrt et al., 2016) that investigated how the elt-2 gene is controlled by END-1, END-3 and ELT-7, the 3 endoderm specific GATA factors that lie upstream in the regulatory hierarchy. We also discuss the unexpected result that ELT-2, if expressed sufficiently early and at sufficiently high levels, can specify the C. elegans endoderm, replacing the normal functions of END-1 and END-3.
RESUMO
The Caenorhabditis elegans vitellogenin genes are transcribed in the intestine of adult hermaphrodites but not of males. A 44-bp region from the vit-2 gene promoter is able largely to reconstitute this tissue-, stage- and sex-specific-expression. This "enhancer" contains a binding site for the DM-domain factor MAB-3, the male-specific repressor of vitellogenesis, as well as an activator site that we show is the direct target of the intestinal GATA factor ELT-2. We further show that the enhancer is directly activated by the winged-helix/forkhead-factor FKH-9, (whose gene has been shown by others to be a direct target of DAF-16), by an unknown activator binding to the MAB-3 site, and by the full C. elegans TGF-ß/Sma/Mab pathway acting within the intestine. The vit-2 gene has been shown by others to be repressed by the daf-2/daf-16 insulin signaling pathway, which so strongly influences aging and longevity in C. elegans. We show that the activity of the 44 bp vit-2 enhancer is abolished by loss of daf-2 but is restored by simultaneous loss of daf-16. DAF-2 acts from outside of the intestine but DAF-16 acts both from outside of the intestine and from within the intestine where it binds directly to the same non-canonical target site that interacts with FKH-9. Activity of the 44 bp vit-2 enhancer is also inhibited by loss of the germline, in a manner that is only weakly influenced by DAF-16 but that is strongly influenced by KRI-1, a key downstream effector in the pathway by which germline loss increases C. elegans lifespan. The complex behavior of this enhancer presumably allows vitellogenin gene transcription to adjust to demands of body size, germline proliferation and nutritional state but we suggest that the apparent involvement of this enhancer in aging and longevity "pathways" could be incidental.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Intestinos/embriologia , Vitelogeninas/metabolismo , Animais , Tamanho Corporal , Caenorhabditis elegans , Proliferação de Células , Elementos Facilitadores Genéticos , Feminino , Proteínas de Fluorescência Verde/metabolismo , Insulina/metabolismo , Masculino , Regiões Promotoras Genéticas , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
ELT-2 is the major regulator of genes involved in differentiation, maintenance and function of C. elegans intestine from the early embryo to mature adult. elt-2 responds to overexpression of the GATA transcription factors END-1 and END-3, which specify the intestine, as well as to overexpression of the two GATA factors that are normally involved in intestinal differentiation, ELT-7 and ELT-2 itself. Little is known about the molecular mechanisms underlying these interactions, how ELT-2 levels are maintained throughout development or how such systems respond to developmental perturbations. Here, we analyse elt-2 gene regulation through transgenic reporter assays, ELT-2 ChIP and characterisation of in vitro DNA-protein interactions. Our results indicate that elt-2 is controlled by three discrete regulatory regions conserved between C. elegans and C. briggsae that span >4â kb of 5' flanking sequence. These regions are superficially interchangeable but have quantitatively different enhancer properties, and their combined activities indicate inter-region synergies. Their regulatory activity is mediated by a small number of conserved TGATAA sites that are largely interchangeable and interact with different endodermal GATA factors with only modest differences in affinity. The redundant molecular mechanism that forms the elt-2 regulatory network is robust and flexible, as loss of end-3 halves ELT-2 levels in the early embryo but levels fully recover by the time of hatching. When ELT-2 is expressed under the control of end-1 regulatory elements, in addition to its own endogenous promoter, it can replace the complete set of endoderm-specific GATA factors: END-1, END-3, ELT-7 and (the probably non-functional) ELT-4. Thus, in addition to controlling gene expression during differentiation, ELT-2 is capable of specifying the entire C. elegans endoderm.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Endoderma/embriologia , Endoderma/metabolismo , Fatores de Transcrição GATA/genética , Regulação da Expressão Gênica no Desenvolvimento , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Sequência Conservada , DNA/metabolismo , Fatores de Transcrição GATA/metabolismo , Redes Reguladoras de Genes , Mucosa Intestinal/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Germ cells in animals are highly specialized to preserve the genome. A distinct set of chromatin structures must be properly established in germ cells to maintain cell fate and genome integrity. We describe DNA-surface interactions in activated Caenorhabditis elegans oocytes that are revealed through the activity of an endogenous nuclease ('endocleavage'). RESULTS: Our analysis began with an unexpected observation that a majority (>50%) of DNA from ovulated but unfertilized C. elegans oocytes can be recovered in fragments of approximately 500 base pairs or shorter, cleaved at regular intervals (10 to 11 nt) along the DNA helix. In some areas of the genome, DNA cleavage patterns in these endoreduplicated oocytes appear consistent from cell-to-cell, indicating coherent rotational positioning of the DNA in chromatin. Particularly striking in this analysis are arrays of sensitive sites with a periodicity of approximately 10 bp that persist for several hundred base pairs of genomic DNA, longer than a single nucleosome core. Genomic regions with a strong bias toward a 10-nt periodic occurrence of A(n)/T(n) (so-called PATC regions) appear to exhibit a high degree of rotational constraint in endocleavage phasing, with a strong tendency for the periodic A(n)/T(n) sites to remain on the face of the helix protected from nuclease digestion. CONCLUSION: The present analysis provides evidence for an unusual structure in C. elegans oocytes in which genomic DNA and associated protein structures are coherently linked.
RESUMO
The transcriptional regulatory hierarchy that controls development of the Caenorhabditis elegans endoderm begins with the maternally provided SKN-1 transcription factor, which determines the fate of the EMS blastomere of the four-cell embryo. EMS divides to produce the posterior E blastomere (the clonal progenitor of the intestine) and the anterior MS blastomere, a major contributor to mesoderm. This segregation of lineage fates is controlled by an intercellular signal from the neighboring P2 blastomere and centers on the HMG protein POP-1. POP-1 would normally repress the endoderm program in both E and MS but two consequences of the P2-to-EMS signal are that POP-1 is exported from the E-cell nucleus and the remaining POP-1 is converted to an endoderm activator by complexing with SYS-1, a highly diverged ß-catenin. In the single E cell, a pair of genes encoding small redundant GATA-type transcription factors, END-1 and END-3, are transcribed under the combined control of SKN-1, the POP-1/SYS-1 complex, as well as the redundant pair of MED-1/2 GATA factors, themselves direct zygotic targets of SKN-1 in the EMS cell. With the expression of END-1/END-3, the endoderm is specified. END-1 and END-3 then activate transcription of a further set of GATA-type transcription factors that drive intestine differentiation and function. One of these factors, ELT-2, appears predominant; a second factor, ELT-7, is partially redundant with ELT-2. The mature intestine expresses several thousand genes, apparently all controlled, at least in part, by cis-acting GATA-type motifs.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Intestinos/crescimento & desenvolvimento , Animais , Blastômeros/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Mucosa Intestinal/metabolismo , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Proteínas de Ligação a RNA , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Erlins are highly conserved proteins associated with lipid rafts within the endoplasmic reticulum (ER). Biochemical studies in mammalian cell lines have shown that erlins are required for ER associated protein degradation (ERAD) of activated inositol-1,4,5-trisphosphate receptors (IP3Rs), implying that erlin proteins might negatively regulate IP3R signalling. In humans, loss of erlin function appears to cause progressive intellectual disability, motor dysfunction and joint contractures. However, it is unknown if defects in IP3R ERAD are the underlying cause of this disease phenotype, whether ERAD of activated IP3Rs is the only function of erlin proteins, and what role ERAD plays in regulating IP3R-dependent processes in the context of an intact animal or embryo. In this study, we characterize the erlin homologue of the nematode Caenorhabditis elegans and examine erlin function in vivo. We specifically set out to test whether C. elegans erlin modulates IP3R-dependent processes, such as egg laying, embryonic development and defecation rates. We also explore the possibility that erlin might play a more general role in the ERAD pathway of C. elegans. RESULTS: We first show that the C. elegans erlin homologue, ERL-1, is highly similar to mammalian erlins with respect to amino acid sequence, domain structure, biochemical properties and subcellular location. ERL-1 is present throughout the C. elegans embryo; in adult worms, ERL-1 appears restricted to the germline. The expression pattern of ERL-1 thus only partially overlaps with that of ITR-1, eliminating the possibility of ERL-1 being a ubiquitous and necessary regulator of ITR-1. We show that loss of ERL-1 does not affect overall phenotype, or alter brood size, embryonic development or defecation cycle length in either wild type or sensitized itr-1 mutant animals. Moreover we show that ERL-1 deficient worms respond normally to ER stress conditions, suggesting that ERL-1 is not an essential component of the general ERAD pathway. CONCLUSIONS: Although loss of erlin function apparently causes a strong phenotype in humans, no such effect is seen in C. elegans. C. elegans erlin does not appear to be a ubiquitous major modulator of IP3 receptor activity nor does erlin appear to play a major role in ERAD.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Degradação Associada com o Retículo Endoplasmático/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Defecação/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/fisiologia , Degradação Associada com o Retículo Endoplasmático/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Dados de Sequência Molecular , Mutação/genética , FenótipoRESUMO
Budovskaya et al. (Cell. 134, 291-303, 2008) have proposed that the ELT-3 GATA factor regulates somatic aging genes, including those expressed in the intestine, and participates in a transcription factor circuit that "guides Caenorhabditis elegans aging". We have re-investigated two key features of this proposal: (i) expression of elt-3 in the C. elegans adult intestine where the majority of somatic aging genes are expressed, and; (ii) the ability of elt-3 loss-of-function to revert the extended lifespan of daf-2(e1370) mutants. We find that: (i) in agreement with our previously published results, ELT-3 expression is largely hypodermal and is not expressed at significant levels in the adult C. elegans intestine, and; (ii) the elt-3(vp1) zinc-finger deletion mutant does not significantly influence the extended lifespan of daf-2(e1370) mutants. We thus suggest that the role of ELT-3 in C. elegans aging should be re-evaluated.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , AnimaisRESUMO
Oocytes were purified from the temperature-sensitive fertilization-defective fer-1(b232ts) mutant of the nematode Caenorhabditis elegans and used for comprehensive mass spectrometric analysis. Using stringent criteria, 1165 C. elegans proteins were identified; at lower stringency, an additional 288 proteins were identified. We validate the high degree of sample purity and evaluate several possible sources of bias in the proteomic data. We compare the classes of proteins identified in the current oocyte proteome with protein classes identified in our previously determined oocyte transcriptome. The oocyte proteome appears enriched in proteins likely to be needed immediately upon fertilization, whereas the transcriptome appears enriched in molecules and processes needed later in embryogenesis. The current study provides fundamental background information for future more detailed studies of oocyte biology.
Assuntos
Caenorhabditis elegans/metabolismo , Oócitos/metabolismo , Proteoma/análise , Proteômica/métodos , Animais , Feminino , Espectrometria de Massas/métodosRESUMO
The C. elegans digestive tract (pharynx, intestine, and rectum) contains only approximately 100 cells but develops under the control of the same types of transcription factors (e.g. FoxA and GATA factors) that control digestive tract development in far more complex animals. The GATA-factor dominated core regulatory hierarchy directing development of the homogenous clonal intestine from oocyte to mature organ is now known with some degree of certainty, setting the stage for more biochemical experiments to understand developmental mechanisms. The FoxA-factor dominated development of the pharynx (and rectum) is less well understood but is beginning to reveal how transcription factor combinations produce unique cell types within organs.
Assuntos
Caenorhabditis elegans/embriologia , Trato Gastrointestinal/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Diferenciação Celular/genética , Linhagem da Célula , Desenvolvimento Embrionário/genética , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Fatores de Transcrição GATA/fisiologia , Trato Gastrointestinal/citologia , Intestinos/citologia , Intestinos/embriologia , Faringe/citologia , Faringe/embriologia , Reto/citologia , Reto/embriologia , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologiaRESUMO
We review recent studies that have advanced our understanding of the molecular mechanisms regulating transcription in the nematode C. elegans. Topics covered include: (i) general properties of C. elegans promoters; (ii) transcription factors and transcription factor combinations involved in cell fate specification and cell differentiation; (iii) new roles for general transcription factors; (iv) nucleosome positioning in C. elegans "chromatin"; and (v) some characteristics of histone variants and histone modifications and their possible roles in controlling C. elegans transcription.
Assuntos
Regulação da Expressão Gênica , Transcrição Gênica , Animais , Caenorhabditis elegans/genética , Linhagem da Célula/genética , Regiões Promotoras Genéticas , Fatores de TranscriçãoRESUMO
Starting with SAGE-libraries prepared from C. elegans FAC-sorted embryonic intestine cells (8E-16E cell stage), from total embryos and from purified oocytes, and taking advantage of the NextDB in situ hybridization data base, we define sets of genes highly expressed from the zygotic genome, and expressed either exclusively or preferentially in the embryonic intestine or in the intestine of newly hatched larvae; we had previously defined a similarly expressed set of genes from the adult intestine. We show that an extended TGATAA-like sequence is essentially the only candidate for a cis-acting regulatory motif common to intestine genes expressed at all stages. This sequence is a strong ELT-2 binding site and matches the sequence of GATA-like sites found to be important for the expression of every intestinal gene so far analyzed experimentally. We show that the majority of these three sets of highly expressed intestinal-specific/intestinal-enriched genes respond strongly to ectopic expression of ELT-2 within the embryo. By flow-sorting elt-2(null) larvae from elt-2(+) larvae and then preparing Solexa/Illumina-SAGE libraries, we show that the majority of these genes also respond strongly to loss-of-function of ELT-2. To test the consequences of loss of other transcription factors identified in the embryonic intestine, we develop a strain of worms that is RNAi-sensitive only in the intestine; however, we are unable (with one possible exception) to identify any other transcription factor whose intestinal loss-of-function causes a phenotype of comparable severity to the phenotype caused by loss of ELT-2. Overall, our results support a model in which ELT-2 is the predominant transcription factor in the post-specification C. elegans intestine and participates directly in the transcriptional regulation of the majority (>80%) of intestinal genes. We present evidence that ELT-2 plays a central role in most aspects of C. elegans intestinal physiology: establishing the structure of the enterocyte, regulating enzymes and transporters involved in digestion and nutrition, responding to environmental toxins and pathogenic infections, and regulating the downstream intestinal components of the daf-2/daf-16 pathway influencing aging and longevity.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Intestinos/fisiologia , Animais , Sequência de Bases , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Biologia Computacional , Fatores de Transcrição GATA/genética , Intestinos/anatomia & histologia , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The intestine is one of the major organs in C. elegans and is largely responsible for food digestion and assimilation as well as the synthesis and storage of macromolecules. In addition, the intestine is emerging as a powerful experimental system in which to study such universal biological phenomena as vesicular trafficking, biochemical clocks, stress responses and aging. The present chapter describes some of these many and varied properties of the C. elegans intestine: the embryonic cell lineage, intestine morphogenesis, structure and physiology of the intestinal cell and, finally, the transcription factor network controlling intestine development and function.
Assuntos
Caenorhabditis elegans/anatomia & histologia , Animais , Caenorhabditis elegans/fisiologia , Humanos , Intestinos/anatomia & histologia , Intestinos/fisiologiaRESUMO
A SAGE library was prepared from hand-dissected intestines from adult Caenorhabditis elegans, allowing the identification of >4000 intestinally-expressed genes; this gene inventory provides fundamental information for understanding intestine function, structure and development. Intestinally-expressed genes fall into two broad classes: widely-expressed "housekeeping" genes and genes that are either intestine-specific or significantly intestine-enriched. Within this latter class of genes, we identified a subset of highly-expressed highly-validated genes that are expressed either exclusively or primarily in the intestine. Over half of the encoded proteins are candidates for secretion into the intestinal lumen to hydrolyze the bacterial food (e.g. lysozymes, amoebapores, lipases and especially proteases). The promoters of this subset of intestine-specific/intestine-enriched genes were analyzed computationally, using both a word-counting method (RSAT oligo-analysis) and a method based on Gibbs sampling (MotifSampler). Both methods returned the same over-represented site, namely an extended GATA-related sequence of the general form AHTGATAARR, which agrees with experimentally determined cis-acting control sequences found in intestine genes over the past 20 years. All promoters in the subset contain such a site, compared to <5% for control promoters; moreover, our analysis suggests that the majority (perhaps all) of genes expressed exclusively or primarily in the worm intestine are likely to contain such a site in their promoters. There are three zinc-finger GATA-type factors that are candidates to bind this extended GATA site in the differentiating C. elegans intestine: ELT-2, ELT-4 and ELT-7. All evidence points to ELT-2 being the most important of the three. We show that worms in which both the elt-4 and the elt-7 genes have been deleted from the genome are essentially wildtype, demonstrating that ELT-2 provides all essential GATA-factor functions in the intestine. The SAGE analysis also identifies more than a hundred other transcription factors in the adult intestine but few show an RNAi-induced loss-of-function phenotype and none (other than ELT-2) show a phenotype primarily in the intestine. We thus propose a simple model in which the ELT-2 GATA factor directly participates in the transcription of all intestine-specific/intestine-enriched genes, from the early embryo through to the dying adult. Other intestinal transcription factors would thus modulate the action of ELT-2, depending on the worm's nutritional and physiological needs.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Fatores de Transcrição GATA/fisiologia , Modelos Genéticos , Transcrição Gênica , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Fatores de Transcrição GATA/genética , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Regiões Promotoras GenéticasRESUMO
The med-1 and med-2 genes encode small, highly similar proteins related to GATA-type transcription factors and have been proposed as necessary for specification of both the mesoderm and the endoderm of Caenorhabditis elegans. However, we have previously presented evidence that neither maternal nor zygotic expression of the med-1/2 genes is necessary to specify the C. elegans endoderm. Contradicting our conclusions, a recent report presented evidence, based on presumed transgene-induced cosuppression, that the med-1/2 genes do indeed show an endoderm-specifying maternal effect. In this article, we reinvestigate med-2(-); med-1(-) embryos using a med-2- specific null allele instead of the chromosomal deficiences used previously and confirm our previous results: the large majority (approximately 84%) of med-2(-); med-1(-) embryos express gut granules. We also reinvestigate the possibility of a maternal med-1/2 effect by direct injection of med dsRNA into sensitized (med-deficient) hermaphrodites using the standard protocol known to be effective in ablating maternal transcripts, but again find no evidence for any significant maternal med-1/2 effect. We do, however, show that expression of gut granules in med-1/2-deficient embryos is exquisitely sensitive to RNAi against the vacuolar ATPase-encoding unc-32 gene [present on the same multicopy med-1(+)-containing transgenic balancer used in support of the maternal med-1/2 effect]. We thus suggest that the experimental evidence for a maternal med-1/2 effect should be reexamined and may instead reflect cosuppression caused by multiple transgenic unc-32 sequences, not med sequences.
Assuntos
Padronização Corporal/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Endoderma/metabolismo , Fatores de Transcrição GATA/genética , Zigoto/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Grânulos Citoplasmáticos/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Interferência de RNA , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in a variety of functions in vertebrates, especially angiogenesis. Here we identify and characterize a PDGF/VEGF-like factor (named PVF-1) from the nematode C. elegans. We show that PVF-1 has biochemical properties similar to vertebrate PDGF/VEGF growth factors. More important, PVF-1 binds to the human receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) and is able to induce angiogenesis in two model systems derived from vertebrates. Our results highlight the widespread evolutionary conservation of this important class of growth factors and raise the possibility that C. elegans can provide a simple experimental system in which to investigate how these factors function.