RESUMO
In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA1,2. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer3. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites4,5. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Simulação por Computador , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Complexo de Reconhecimento de Origem/química , Ligação Proteica , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin.
Assuntos
Cromatina/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas Repressoras/metabolismo , Animais , Cromatina/química , Cromatina/genética , Histonas/metabolismo , Humanos , Modelos Biológicos , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/genética , Domínios Proteicos , Estrutura Terciária de Proteína , Domínios RING Finger , Proteínas Repressoras/química , Proteínas Repressoras/genética , UbiquitinaçãoRESUMO
DNA replication has been reconstituted in vitro with yeast proteins, and the minimal system requires the coordinated assembly of 16 distinct replication factors, consisting of 42 polypeptides. To understand the molecular interplay between these factors at the single residue level, new structural biology tools are being developed. Inspired by advances in single-molecule fluorescence imaging and cryo-tomography, novel single-particle cryo-EM experiments have been used to characterise the structural mechanism for the loading of the replicative helicase. Here, we discuss how in silico reconstitution of single-particle cryo-EM data can help describe dynamic systems that are difficult to approach with conventional three-dimensional classification tools.
Assuntos
Replicação do DNA , Imagem Individual de Molécula , Microscopia Crioeletrônica/métodos , Imagem Individual de Molécula/métodos , TomografiaRESUMO
The loading of the MCM replicative helicase onto eukaryotic origins of replication occurs via a sequential, symmetric mechanism. Here, we describe a method to study this multistep reaction using electron microscopy. Tools presented include protein expression and purification protocols, methods to produce asymmetric replication origin substrates and bespoke image processing strategies. DNA templates include recognisable protein roadblocks that help to orient DNA replication factors along a specific origin sequence. Detailed electron microscopy image processing protocols are provided to reposition 2D averages onto the original micrograph for the in silico reconstitution of fully occupied origins of replication. Using these tools, a chemically trapped helicase loading intermediate is observed sliding along origin DNA, showcasing a key feature of the MCM loading mechanism. Although developed to study replicative helicase loading, this method can be employed to investigate the mechanism of other multicomponent biochemical reactions, occurring on a flexible polymeric substrate.
Assuntos
DNA Helicases , Origem de Replicação , DNA , DNA Helicases/metabolismo , Replicação do DNA , Microscopia EletrônicaRESUMO
Genome duplication is safeguarded by constantly adjusting the activity of the replicative CMG (CDC45-MCM2-7-GINS) helicase. However, minichromosome maintenance proteins (MCMs)-the structural core of the CMG helicase-have never been visualized at sites of DNA synthesis inside a cell (the so-called MCM paradox). Here, we solve this conundrum by showing that anti-MCM antibodies primarily detect inactive MCMs. Upon conversion of inactive MCMs to CMGs, factors that are required for replisome activity bind to the MCM scaffold and block MCM antibody binding sites. Tagging of endogenous MCMs by CRISPR-Cas9 bypasses this steric hindrance and enables MCM visualization at active replisomes. Thus, by defining conditions for detecting the structural core of the replicative CMG helicase, our results explain the MCM paradox, provide visual proof that MCMs are an integral part of active replisomes in vivo, and enable the investigation of replication dynamics in living cells exposed to a constantly changing environment.
Assuntos
Replicação do DNA , Proteínas de Manutenção de Minicromossomo , DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismoRESUMO
Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Quinase do Ponto de Checagem 2/metabolismo , Componente 4 do Complexo de Manutenção de Minicromossomo/química , Componente 4 do Complexo de Manutenção de Minicromossomo/metabolismo , Simulação de Acoplamento Molecular , Nucleotídeos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Especificidade por SubstratoRESUMO
BACKGROUND: Asymmetric arginine dimethylation of histone H4R3 to H4R3me2a by protein arginine methyltransferase 1 (PRMT1) has been implicated to play a key role in gene activation throughout vertebrates. PRMT1 knockout in mouse leads to embryonic lethality. This and the uterus-enclosed nature of the mouse embryo make it difficult to determine the development role of PRMT1 in mammals. METHODS: We took advantage of the external development of the diploid anuran Xenopus tropicalis and adapted the TALEN genome editing technology to knock out PRMT1 in order to investigate how PRMT1 participates in vertebrate development. RESULTS: We observed that PRMT1 knockout had no apparent effect on embryogenesis because normally feeding tadpoles were formed, despite the reduced asymmetric H4R3 di-methylation (H4R3me2a) due to the knockout. However, PRMT1 knockout tadpoles had severely reduced growth even with normal growth hormone gene expression. These tadpoles were also stalled in development shortly after feeding began at stages 44/45 and died within 2 weeks, well before the onset of metamorphosis. In situ analyses revealed broad cessation or drastic reduction in cell proliferation in diverse organs including the eye, brain, spinal cord, liver, and intestine. CONCLUSIONS: Our findings suggest that PRMT1 is not required for embryogenesis but is a key regulator for normal progression of vertebrate development and growth. GENERAL SIGNIFICANCE: The similarities and differences between PRMT1 knockout Xenopus tropicalis and mouse suggest that two distinct phases of vertebrate development: early embryogenesis and subsequent growth/organ maturation, have different but evolutionally conserved requirement for epigenetic modifications.
Assuntos
Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/genética , Arginina/metabolismo , Proliferação de Células , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes/métodos , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Larva/metabolismo , Masculino , Metamorfose Biológica/genética , Metamorfose Biológica/fisiologia , Metilação , Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Ativação Transcricional , Xenopus/genética , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Thyrotropin (TSH), a pituitary glycoprotein hormone that stimulates the thyroid gland, has been cloned and sequenced from over a dozen teleost fish species. Although TSH is established as a primary driver of systemic thyroid status in mammals, its importance in the regulation of fish thyroid function is still uncertain. We review recent studies indicating that TSH structure is highly conserved across species representing six teleost families. These studies have found TSH messenger RNA consistently expressed in teleost pituitary tissue, although ectopic expression, particularly in gonads, has also been observed. They have also provided evidence for negative feedback inhibition of TSH expression by thyroid hormones, as well as stimulation by hypothalamic peptides. Descriptive studies have found increased TSHbeta expression associated with life history events thought to be promoted by thyroid hormones. These results, coupled with the discovery of a G-protein coupled TSH receptor in several teleost species, supports an active and conserved role for TSH in the regulation of teleost thyroid function. The relative importance of central pathways in regulating thyroid hormone provision to targets and the identity of a proposed thyrotropin-inhibiting factor in teleost fish are still unanswered questions whose resolution will be facilitated by development of methods to measure circulating TSH and its secretion from the pituitary gland.
Assuntos
Peixes/genética , Tireotropina/genética , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Hipófise/metabolismo , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Alinhamento de Sequência , Tireotropina/química , Tireotropina/metabolismo , Tireotropina Subunidade beta/química , Tireotropina Subunidade beta/genéticaRESUMO
Ubiquitin C-terminal hydrolase deubiquitinase BAP1 is an essential tumor suppressor involved in cell growth control, DNA damage response, and transcriptional regulation. As part of the Polycomb repression machinery, BAP1 is activated by the deubiquitinase adaptor domain of ASXL1 mediating gene repression by cleaving ubiquitin (Ub) from histone H2A in nucleosomes. The molecular mechanism of BAP1 activation by ASXL1 remains elusive, as no structures are available for either BAP1 or ASXL1. Here, we present the crystal structure of the BAP1 ortholog from Drosophila melanogaster, named Calypso, bound to its activator, ASX, homolog of ASXL1. Based on comparative structural and functional analysis, we propose a model for Ub binding by Calypso/ASX, uncover decisive structural elements responsible for ASX-mediated Calypso activation, and characterize the interaction with ubiquitinated nucleosomes. Our results give molecular insight into Calypso function and its regulation by ASX and provide the opportunity for the rational design of mechanism-based therapeutics to treat human BAP1/ASXL1-related tumors.
Assuntos
Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Drosophila/química , Drosophila melanogaster/química , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Ubiquitina/metabolismoRESUMO
The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc-Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad-Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development.
Assuntos
Células-Tronco Adultas/metabolismo , Apoptose/fisiologia , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Intestinos/embriologia , Metamorfose Biológica/fisiologia , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas de Xenopus/biossíntese , Animais , Proteínas de Ligação a DNA/genética , Larva/genética , Larva/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Smad1 , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Various animal models are indispensible in biomedical research. Increasing awareness and regulations have prompted theadaptation of more humane approaches in the use of laboratory animals. With the development of easier and faster methodologies to generate genetically altered animals, convenient and humane methods to genotype these animals are important for research involving such animals. Here, we report skin swabbing as a simple and noninvasive method for extracting genomic DNA from mice and frogs for genotyping. We show that this method is highly reliable and suitable for both immature and adult animals. Our approach allows a simpler and more humane approach for genotyping vertebrate animals.
RESUMO
Various animal models are indispensible in biomedical research. Increasing awareness and regulations have prompted the adaptation of more humane approaches in the use of laboratory animals. With the development of easier and faster methodologies to generate genetically altered animals, convenient and humane methods to genotype these animals are important for research involving such animals. Here, we report skin swabbing as a simple and noninvasive method for extracting genomic DNA from mice and frogs for genotyping. We show that this method is highly reliable and suitable for both immature and adult animals. Our approach allows a simpler and more humane approach for genotyping vertebrate animals.
Assuntos
Animais de Laboratório/genética , DNA/genética , Genótipo , Pele , Animais , Pesquisa Biomédica , CamundongosRESUMO
Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain-DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition.
Assuntos
Proteínas Nucleares/química , Nucleossomos/química , Acetilação , Sequência de Aminoácidos , Histonas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis.
Assuntos
Células-Tronco Adultas/metabolismo , Amidoidrolases/metabolismo , Intestinos/citologia , Metamorfose Biológica , Tri-Iodotironina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Amidoidrolases/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Intestinos/fisiologia , Camundongos , Dados de Sequência Molecular , Elementos de Resposta , Receptores X de Retinoides/metabolismo , Proteínas de Xenopus/genéticaRESUMO
BACKGROUND: The thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells. RESULTS: Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis. CONCLUSIONS: Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).
RESUMO
BACKGROUND: Intestinal remodeling during amphibian metamorphosis has long been studied as a model for the formation of the adult organs in vertebrates, especially the formation of adult organ-specific stem cells. Like all other processes during metamorphosis, this process is controlled by thyroid hormone (T3), which affects cell fate and behavior through transcriptional regulation of target genes by binding to T3 receptors (TRs). Earlier studies have shown that Sonic hedgehog (Shh) is induced by T3 in the developing adult stem cells and that the Shh receptor and other downstream components are present in the connective tissue and at lower levels in the muscles at the climax of intestinal remodeling. However, no in vivo studies have carried out to investigate whether Shh produced in the adult cells can regulate the connective tissue to promote intestinal maturation. RESULTS: We have addressed this issue by treating tadpoles with Shh inhibitor cyclopamine. We showed that cyclopamine but not the structurally related chemical tomatidine inhibited the expression of Shh response genes BMP4, Snai2, and Twist1. More importantly, we showed that cyclopamine reduced the cell proliferation of both the developing adult stem cells as well as cells in the other intestinal tissues at the climax of metamorphosis, leading to delayed/incomplete remodeling of the intestine at the end of metamorphosis. We further revealed that both Snai2 and Twist1 were strongly upregulated during metamorphosis in the intestine and their expression was restricted to the connective tissue. CONCLUSIONS: Our results suggest that Shh indeed signals the connective tissue whereby it can increase adult stem cell proliferation and promote formation of the adult intestine.
RESUMO
Methods were evaluated to quantify sclerotia in rice fields affected with aggregate sheath spot disease. Recovered sclerotia were compared with disease ratings at harvest for paddies subjected to various postharvest cultural straw management practices. Sclerotial inoculum level was not always correlated with subsequent disease incidence in rice aggregate sheath spot disease, suggesting alternative sources of inoculum and other factors influencing disease development. Straw management practices affected the inoculum load of Rhizoctonia oryzae-sativae, as determined by methods recovering sclerotia from California rice field soil samples. Traditional wet sieving of soil samples was improved using a potassium carbonate solution to float heavier sclerotia that normally remained in the sediment. The relationship of inoculum in the soil from seedbeds and the incidence of resulting disease measured just before harvest was investigated over 3 years at three sites. A linear dose-response was found at only one site where the number of floating sclerotia was more strongly correlated with disease incidence than total number of sclerotia (floating plus nonfloating sclerotia). Floating sclerotia were more easily counted using a water floatation extraction (WFE) than were total sclerotia by using a potassium carbonate floatation extraction (PCFE) or a combination of the two assays (WFE + PCFE), and the WFE more accurately predicted disease incidence than did either PCFE or WFE + PCFE. All assays detected significant differences between inoculum levels as influenced by various straw residue management practices, with removal of straw residue significantly reducing number of soil-borne sclerotia.
RESUMO
The Pygo-BCL9 complex is a chromatin reader, facilitating ß-catenin-mediated oncogenesis, and is thus emerging as a potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygo's PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from the BCL9 HD1 domain. Here, we report the first use of fragment-based screening by NMR to identify small molecules that block protein-protein interactions by a PHD finger. This led to the discovery of a set of benzothiazoles that bind to a cleft emanating from the PHD-HD1 interface, as defined by X-ray crystallography. Furthermore, we discovered a benzimidazole that docks into the H3K4me specificity pocket and displaces the native H3K4me peptide from the PHD finger. Our study demonstrates the ligandability of the Pygo-BCL9 complex and uncovers a privileged scaffold as a template for future development of lead inhibitors of oncogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Antineoplásicos/química , Benzimidazóis/química , Benzotiazóis/química , Histonas/química , Proteínas de Neoplasias/química , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Ligação Competitiva , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , Descoberta de Drogas , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de TranscriçãoRESUMO
The mammalian intestine has long been used as a model to study organ-specific adult stem cells, which are essential for organ repair and tissue regeneration throughout adult life. The establishment of the intestinal epithelial cell self-renewing system takes place during perinatal development when the villus-crypt axis is established with the adult stem cells localized in the crypt. This developmental period is characterized by high levels of plasma thyroid hormone (T3) and T3 deficiency is known to impair intestinal development. Determining how T3 regulates adult stem cell development in the mammalian intestine can be difficult due to maternal influences. Intestinal remodeling during amphibian metamorphosis resembles perinatal intestinal maturation in mammals and its dependence on T3 is well established. A major advantage of the amphibian model is that it can easily be controlled by altering the availability of T3. The ability to manipulate and examine this relatively rapid and localized formation of adult stem cells has greatly assisted in the elucidation of molecular mechanisms regulating their formation and further revealed evidence that supports conservation in the underlying mechanisms of adult stem cell development in vertebrates. Furthermore, genetic studies in Xenopus laevis indicate that T3 actions in both the epithelium and the rest of the intestine, most likely the underlying connective tissue, are required for the formation of adult stem cells. Molecular analyses suggest that cell-cell interactions involving hedgehog and BMP pathways are critical for the establishment of the stem cell niche that is essential for the formation of the adult intestinal stem cells.
RESUMO
BACKGROUND: Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells. METHODOLOGY/PRINCIPAL FINDINGS: The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium. CONCLUSIONS/SIGNIFICANCE: Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells.