RESUMO
The regulation of floral organ identity was investigated using a forward genetic approach in five floral homeotic mutants of Thalictrum, a noncore eudicot. We hypothesized that these mutants carry defects in the floral patterning genes. Mutant characterization comprised comparative floral morphology and organ identity gene expression at early and late developmental stages, followed by sequence analysis of coding and intronic regions to identify transcription factor binding sites and protein-protein interaction (PPI) motifs. Mutants exhibited altered expression of floral MADS-box genes, which further informed the function of paralogs arising from gene duplications not found in reference model systems. The ensuing modified BCE models for the mutants supported instances of neofunctionalization (e.g., B-class genes expressed ectopically in sepals), partial redundancy (E-class), or subfunctionalization (C-class) of paralogs. A lack of deleterious mutations in the coding regions of candidate floral MADS-box genes suggested that cis-regulatory or trans-acting mutations are at play. Consistent with this hypothesis, double-flower mutants had transposon insertions or showed signs of transposon activity in the regulatory intron of AGAMOUS (AG) orthologs. Single amino acid substitutions were also found, yet they did not fall on any of the identified DNA binding or PPI motifs. In conclusion, we present evidence suggesting that transposon activity and regulatory mutations in floral homeotic genes likely underlie the striking phenotypes of these Thalictrum floral homeotic mutants.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Animais , Flores/genética , Genes Homeobox , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Variation in regulatory DNA is thought to drive phenotypic variation, evolution, and disease. Prior studies of regulatory DNA and transcription factors across animal species highlighted a fundamental conundrum: Transcription factor binding domains and cognate binding sites are conserved, while regulatory DNA sequences are not. It remains unclear how conserved transcription factors and dynamic regulatory sites produce conserved expression patterns across species. Here, we explore regulatory DNA variation and its functional consequences within Arabidopsis thaliana, using chromatin accessibility to delineate regulatory DNA genome-wide. Unlike in previous cross-species comparisons, the positional homology of regulatory DNA is maintained among A. thaliana ecotypes and less nucleotide divergence has occurred. Of the â¼50,000 regulatory sites in A. thaliana, we found that 15% varied in accessibility among ecotypes. Some of these accessibility differences were associated with extensive, previously unannotated sequence variation, encompassing many deletions and ancient hypervariable alleles. Unexpectedly, for the majority of such regulatory sites, nearby gene expression was unaffected. Nevertheless, regulatory sites with high levels of sequence variation and differential chromatin accessibility were the most likely to be associated with differential gene expression. Finally, and most surprising, we found that the vast majority of differentially accessible sites show no underlying sequence variation. We argue that these surprising results highlight the necessity to consider higher-order regulatory context in evaluating regulatory variation and predicting its phenotypic consequences.
Assuntos
Arabidopsis/genética , Ecótipo , Elementos Reguladores de Transcrição , Arabidopsis/metabolismo , Sequência de Bases , Desoxirribonuclease I , Variação Estrutural do Genoma , Análise de Sequência de DNARESUMO
In the model plant Arabidopsis thaliana, a core eudicot, the floral homeotic C-class gene AGAMOUS (AG) has a dual role specifying reproductive organ identity and floral meristem determinacy. We conduct a functional analysis of the putative AG ortholog ThtAG1 from the ranunculid Thalictrum thalictroides, a representative of the sister lineage to all other eudicots. Down-regulation of ThtAG1 by virus-induced gene silencing resulted in homeotic conversion of stamens and carpels into sepaloid organs and loss of flower determinacy. Moreover, flowers exhibiting strong silencing of ThtAG1 phenocopied the double-flower ornamental cultivar T. thalictroides 'Double White.' Molecular analysis of 'Double White' ThtAG1 alleles revealed the insertion of a retrotransposon causing either nonsense-mediated decay of transcripts or alternative splicing that results in mutant proteins with K-domain deletions. Biochemical analysis demonstrated that the mutation abolishes protein-protein interactions with the putative E-class protein ThtSEP3. C- and E-class protein heterodimerization is predicted by the floral quartet model, but evidence for the functional importance of this interaction is scarce outside the core eudicots. Our findings therefore corroborate the importance and conservation of the interactions between C- and E-class proteins. This study provides a functional description of a full C-class mutant in a noncore ("basal") eudicot, an ornamental double flower, affecting both organ identity and meristem determinacy. Using complementary forward and reverse genetic approaches, this study demonstrates deep conservation of the dual C-class gene function and of the interactions between C- and E-class proteins predicted by the floral quartet model.
Assuntos
Regulação da Expressão Gênica de Plantas , Mutação , Thalictrum/metabolismo , Sequência de Aminoácidos , Bioquímica/métodos , Elementos de DNA Transponíveis/genética , Flores , Inativação Gênica , Modelos Genéticos , Dados de Sequência Molecular , Fenótipo , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , RNA/genética , Homologia de Sequência de Aminoácidos , Sequências Repetidas TerminaisRESUMO
BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.
Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Animais , Cães , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos de Linfócitos T , Leucócitos Mononucleares , Distribuição Tecidual , Engenharia Celular/métodosRESUMO
The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of polysaccharides. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation (4 and 7 DPA), enriching for seed coat cells with the INTACT method. We found over 3,000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with 7-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, plant regulatory landscapes from different tissues, cell types, or developmental stages were more dynamic than those generated from bulk tissue in response to environmental perturbations, highlighting the importance of extending studies of regulatory DNA to single tissues and cell types during development.
RESUMO
Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate â¼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Mapeamento Cromossômico , Códon , Desoxirribonuclease I/metabolismo , Éxons , Redes Reguladoras de Genes , Genoma de Planta , Estudo de Associação Genômica Ampla , Luz , Desenvolvimento Vegetal/genética , Ligação Proteica , Elementos Reguladores de Transcrição/genética , Plântula/genética , Fatores de Transcrição/metabolismoRESUMO
Robustness, the ability of organisms to buffer phenotypes against perturbations, has drawn renewed interest among developmental biologists and geneticists. A growing body of research supports an important role of robustness in the genotype to phenotype translation, with far-reaching implications for evolutionary processes and disease susceptibility. Similar to animals and fungi, plant robustness is a function of genetic network architecture. Most perturbations are buffered; however, perturbation of network hubs destabilizes many traits. Here, we review recent advances in identifying molecular robustness mechanisms in plants that have been enabled by a combination of classical genetics and population genetics with genome-scale data.
Assuntos
Redes Reguladoras de Genes , Desenvolvimento Vegetal , Plantas/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Evolução Molecular , Genômica , Genótipo , Meristema , Mutação , Fenótipo , PlântulaRESUMO
Sexual systems are highly variable in flowering plants and an important contributor to floral diversity. The ranunculid genus Thalictrum is especially well-suited to study evolutionary transitions in sexual systems. Homeotic transformation of sexual organs (stamens and carpels) is a plausible mechanism for the transition from hermaphroditic to unisexual flowers in this lineage because flowers of dioecious species develop unisexually from inception. The single-copy gene PISTILLATA (PI) constitutes a likely candidate for rapid switches between stamen and carpel identity. Here, we first characterized the expression pattern of all B class genes in the dioecious species T. dioicum. As expected, all B class orthologs are expressed in stamens from the earliest stages. Certain AP3 lineages were also expressed late in sepal development. We then tested whether orthologs of PI could potentially control sexual system transitions in Thalictrum, by knocking-down their expression in T. dioicum and the hermaphroditic species T. thalictroides. In T. dioicum, we found that ThdPI-1/2 silencing caused stamen primordia to develop into carpels, resulting in male to female flower conversions. In T. thalictroides, we found that ThtPI silencing caused stamen primordia to develop into supernumerary carpels, resulting in hermaphroditic to female flower conversions. These phenotypes illustrate the ability for homeotic mutations to bring about sudden and potentially adaptive changes by altering the function of a single gene. We propose a two-step evolutionary model where transitions from hermaphroditic to unisexual plants in Thalictrum result from two independent mutations at a B class gene locus. Our PI knockdown experiments in T. thalictroides recapitulate the second step in this model: the evolution of female plants as a result of a loss-of-function mutation in a B class gene.