RESUMO
The flower is a hallmark feature that has contributed to the evolutionary success of land plants. Diverse mutagenic agents have been employed as a tool to genetically perturb flower development and identify genes involved in floral patterning and morphogenesis. Since the initial studies to identify genes governing processes such as floral organ specification, mutagenesis in sensitized backgrounds has been used to isolate enhancers and suppressors to further probe the molecular basis of floral development. Here, we first describe two commonly employed methods for mutagenesis (using ethyl methanesulfonate (EMS) or T-DNAs as mutagens), and then describe three methods for identifying a mutation that leads to phenotypic alterations: traditional map-based cloning, modified high-efficiency thermal asymmetric interlaced PCR (mhiTAIL-PCR), and deep sequencing in the plant model Arabidopsis thaliana.
Assuntos
Arabidopsis , Arabidopsis/genética , Testes Genéticos , Mutação , Mutagênese , MutagênicosRESUMO
Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.
Assuntos
Células Endoteliais , Veias , Morfogênese/genética , Artérias , Movimento CelularRESUMO
Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.
Assuntos
Artérias/citologia , Vasos Coronários/citologia , Análise de Célula Única , Células-Tronco/citologia , Células-Tronco/metabolismo , Veias/citologia , Animais , Artérias/metabolismo , Fator II de Transcrição COUP/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Linhagem da Célula , Vasos Coronários/metabolismo , Feminino , Masculino , Camundongos , Análise de Sequência de RNA , Veias/metabolismoRESUMO
UNLABELLED: Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII: C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.
Assuntos
Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Endotélio Vascular/metabolismo , Fator VIII/biossíntese , Regulação da Expressão Gênica/fisiologia , Fator de von Willebrand/biossíntese , Animais , Capilares/citologia , Capilares/metabolismo , Células Endoteliais/citologia , Endotélio Linfático/citologia , Endotélio Vascular/citologia , Feminino , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Fígado/irrigação sanguínea , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Vênulas/citologia , Vênulas/metabolismoRESUMO
Genetic screening has been a powerful tool in identifying new genes in a pathway of interest (forward genetics) or attributing function to a particular gene via mutagenesis (reverse genetics). Small molecule-based chemical genetics is increasingly adapted in Arabidopsis research as a tool for similar purposes, i.e., to identify genes involved in certain biological processes and to dissect the biological roles of a gene. Chemical genetic screens have been successful in circumventing genetic redundancy to assign biological roles to a gene family as well as novel functions for well-known genes. Here, we describe how to screen Arabidopsis seedlings grown on solid medium with chemical compounds.
Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Descoberta de Drogas/métodos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Testes Genéticos , Plântula/efeitos dos fármacos , Plântula/fisiologia , Meios de Cultura , Expressão Gênica , Genes Reporter , Bibliotecas de Moléculas PequenasRESUMO
In Arabidopsis, AUXIN RESPONSE FACTOR 3 (ARF3) belongs to the auxin response factor (ARF) family that regulates the expression of auxin-responsive genes. ARF3 is known to function in leaf polarity specification and gynoecium patterning. In this study, we discovered a previously unknown role for ARF3 in floral meristem (FM) determinacy through the isolation and characterization of a mutant of ARF3 that enhanced the FM determinacy defects of agamous (ag)-10, a weak ag allele. Central players in FM determinacy include WUSCHEL (WUS), a gene critical for FM maintenance, and AG and APETALA2 (AP2), which regulate FM determinacy by repression and promotion of WUS expression, respectively. We showed that ARF3 confers FM determinacy through repression of WUS expression, and associates with the WUS locus in part in an AG-dependent manner. We demonstrated that ARF3 is a direct target of AP2 and partially mediates AP2's function in FM determinacy. ARF3 exhibits dynamic and complex expression patterns in floral organ primordia; altering the patterns spatially compromised FM determinacy. This study uncovered a role for ARF3 in FM determinacy and revealed relationships among genes in the genetic network governing FM determinacy.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Meristema/metabolismo , Proteínas Nucleares/metabolismo , Proteína AGAMOUS de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Meristema/genética , Mutação , Proteínas Nucleares/genética , Plantas Geneticamente ModificadasRESUMO
RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.
Assuntos
Metilação de DNA/genética , DNA Topoisomerases Tipo I/genética , Elementos de DNA Transponíveis/genética , Epigênese Genética , Transcrição Gênica , Arabidopsis/genética , DNA Topoisomerases Tipo I/metabolismo , Inativação Gênica , Histonas/genética , Lisina/genética , Mutação , RNA/genética , RNA Longo não Codificante/genéticaRESUMO
It has been perplexing that DNA topoisomerases, enzymes that release DNA supercoils, play specific roles in development. In this study, using a floral stem cell model in Arabidopsis thaliana, we uncovered a role for TOPOISOMERASE1α (TOP1α) in Polycomb Group (PcG) protein-mediated histone 3 lysine 27 trimethylation (H3K27me3) at, and transcriptional repression of, the stem cell maintenance gene WUSCHEL (WUS). We demonstrated that H3K27me3 deposition at other PcG targets also requires TOP1α. Intriguingly, the repression of some, as well as the expression of many, PcG target genes requires TOP1α. The mechanism that unifies the opposing effects of TOP1α appears to lie in its role in decreasing nucleosome density, which probably allows the binding of factors that either recruit PcG, as we demonstrated for AGAMOUS at the WUS locus, or counteract PcG-mediated regulation. Although TOP1α reduces nucleosome density at all genes, the lack of a 5' nucleosome-free region is a feature that distinguishes PcG targets from nontargets and may condition the requirement for TOP1α for their expression. This study uncovers a connection between TOP1α and PcG, which explains the specific developmental functions of TOP1α.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , DNA Topoisomerases Tipo I/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Nucleossomos/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Proteína AGAMOUS de Arabidopsis/genética , Proteína AGAMOUS de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Diferenciação Celular , DNA Topoisomerases Tipo I/genética , Epigênese Genética , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Meristema/enzimologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Mutação , Brotos de Planta/enzimologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Proteínas do Grupo Polycomb/genética , Transcrição GênicaRESUMO
The flower is a hallmark feature that has contributed to the evolutionary success of land plants. Diverse mutagenic agents have been employed as a tool to genetically perturb flower development and identify genes involved in floral patterning and morphogenesis. Since the initial studies to identify genes governing processes such as floral organ specification, mutagenesis in sensitized backgrounds has been used to isolate enhancers and suppressors to further probe the molecular basis of floral development. Here, we first describe two commonly employed methods for mutagenesis (using ethyl methanesulfonate (EMS) or T-DNAs as mutagens), and then describe three methods for identifying a mutation that leads to phenotypic alterations--traditional map-based cloning, TAIL-PCR, and deep sequencing in the plant model Arabidopsis thaliana.
Assuntos
Arabidopsis/genética , Elementos Facilitadores Genéticos/genética , Flores/genética , Genes Supressores , Mutagênese Insercional/métodos , Mutação , Agrobacterium/genética , Clonagem Molecular , DNA Bacteriano/genética , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Transformação GenéticaRESUMO
BACKGROUND: DNA methylation ensures genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs through a process known as RNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. Once established, DNA methylation at each of the three sequence contexts is maintained through different mechanisms. Although some players involved in RdDM and maintenance methylation have been identified, the underlying molecular mechanisms are not fully understood. To aid the comprehensive identification of players in DNA methylation, we generated a transgenic reporter system that permits genetic and chemical genetic screens in Arabidopsis. RESULTS: A dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal level of luciferase activity, was obtained. LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context. Methylation was present throughout the promoter and LUC coding region. Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Using LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor. CONCLUSION: We developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis. The low basal level of LUCL expression provides an easy readout in genetic and chemical genetic screens that will dissect the mechanisms of RdDM and methylation maintenance.
RESUMO
BACKGROUND: Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis. RESULTS: We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression. CONCLUSION: We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation.
RESUMO
Cell fate specification in development requires transcription factors for proper regulation of gene expression. In Arabidopsis, transcription factors encoded by four classes of homeotic genes, A, B, C and E, act in a combinatorial manner to control proper floral organ identity. The A-class gene APETALA2 (AP2) promotes sepal and petal identities in whorls 1 and 2 and restricts the expression of the C-class gene AGAMOUS (AG) from whorls 1 and 2. However, it is unknown how AP2 performs these functions. Unlike the other highly characterized floral homeotic proteins containing MADS domains, AP2 has two DNA-binding domains referred to as the AP2 domains and its DNA recognition sequence is still unknown. Here, we show that the second AP2 domain in AP2 binds a non-canonical AT-rich target sequence, and, using a GUS reporter system, we demonstrate that the presence of this sequence in the AG second intron is important for the restriction of AG expression in vivo. Furthermore, we show that AP2 binds the AG second intron and directly regulates AG expression through this sequence element. Computational analysis reveals that the binding site is highly conserved in the second intron of AG orthologs throughout Brassicaceae. By uncovering a biologically relevant AT-rich target sequence, this work shows that AP2 domains have wide-ranging target specificities and provides a missing link in the mechanisms that underlie flower development. It also sets the foundation for understanding the basis of the broad biological functions of AP2 in Arabidopsis, as well as the divergent biological functions of AP2 orthologs in dicotyledonous plants.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Flores/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Proteína AGAMOUS de Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Biologia Computacional , Ensaio de Desvio de Mobilidade Eletroforética , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
MicroRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs) impact numerous biological processes in eukaryotes. In addition to biogenesis, turnover contributes to the steady-state levels of small RNAs. One major factor that stabilizes miRNAs and siRNAs in plants as well as siRNAs and piRNAs in animals is 2'-O-methylation on the 3' terminal ribose by the methyltransferase HUA ENHANCER1 (HEN1) [1-6]. Genetic studies with Arabidopsis, Drosophila, and zebrafish hen1 mutants show that 2'-O-methylation protects small RNAs from 3'-to-5' truncation and 3' uridylation, the addition of nontemplated nucleotides, predominantly uridine [2, 7, 8]. Uridylation is a widespread phenomenon that is not restricted to small RNAs in hen1 mutants and is often associated with their reduced accumulation ([7, 9, 10]; reviewed in [11]). The enzymes responsible for 3' uridylation of small RNAs when they lack methylation in plants or animals have remained elusive. Here, we identify the Arabidopsis HEN1 SUPPRESSOR1 (HESO1) gene as responsible for small RNA uridylation in hen1 mutants. HESO1 exhibits terminal nucleotidyl transferase activity, prefers uridine as the substrate nucleotide, and is completely inhibited by 2'-O-methylation. We show that uridylation leads to miRNA degradation, and the degradation is most likely through an enzyme that is distinct from that causing the 3' truncation in hen1 mutants.
Assuntos
Proteínas de Arabidopsis/metabolismo , MicroRNAs/metabolismo , Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , Uridina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metilação , Mutação , Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/genética , RNA Interferente Pequeno/metabolismoRESUMO
Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, we reveal a role of the type III HD-Zip genes, previously known to specify lateral organ polarity, in stem cell termination. Both reduction in HD-Zip expression by over-expression of miR165/166 and mis-expression of HD-Zip genes by rendering them resistant to miR165/166 lead to prolonged floral stem cell activity, indicating that the expression of HD-Zip genes needs to be precisely controlled to achieve floral stem cell termination. We also show that both the ubiquitously expressed ARGONAUTE1 (AGO1) gene and its homolog AGO10, which exhibits highly restricted spatial expression patterns, are required to maintain the correct temporal program of floral stem cells. We provide evidence that AGO10, like AGO1, associates with miR172 and miR165/166 in vivo and exhibits "slicer" activity in vitro. Despite the common biological functions and similar biochemical activities, AGO1 and AGO10 exert different effects on miR165/166 in vivo. This work establishes a network of microRNAs and transcription factors governing the temporal program of floral stem cells and sheds light on the relationships among different AGO genes, which tend to exist in gene families in multicellular organisms.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Flores/citologia , Flores/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Meristema/genética , Meristema/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
The Arabidopsis thaliana transcription factor APETALA2 (AP2) has numerous functions, including roles in seed development, stem cell maintenance, and specification of floral organ identity. To understand the relationship between these different roles, we mapped direct targets of AP2 on a genome-wide scale in two tissue types. We find that AP2 binds to thousands of loci in the developing flower, many of which exhibit AP2-dependent transcription. Opposing, logical effects are evident in AP2 binding to two microRNA genes that influence AP2 expression, with AP2 positively regulating miR156 and negatively regulating miR172, forming a complex direct feedback loop, which also included all but one of the AP2-like miR172 target clade members. We compare the genome-wide direct target repertoire of AP2 with that of SCHLAFMUTZE, a closely related transcription factor that also represses the transition to flowering. We detect clear similarities and important differences in the direct target repertoires that are also tissue specific. Finally, using an inducible expression system, we demonstrate that AP2 has dual molecular roles. It functions as both a transcriptional activator and repressor, directly inducing the expression of the floral repressor AGAMOUS-LIKE15 and directly repressing the transcription of floral activators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1.