RESUMEN
Unraveling the regulatory mechanisms that govern complex traits is pivotal for advancing crop improvement. Here we present a comprehensive regulome atlas for rice (Oryza sativa), charting the chromatin accessibility across 23 distinct tissues from three representative varieties. Our study uncovers 117,176 unique open chromatin regions (OCRs), accounting for ~15% of the rice genome, a notably higher proportion compared to previous reports in plants. Integrating RNA-seq data from matched tissues, we confidently predict 59,075 OCR-to-gene links, with enhancers constituting 69.54% of these associations, including many known enhancer-to-gene links. Leveraging this resource, we re-evaluate genome-wide association study results and discover a previously unknown function of OsbZIP06 in seed germination, which we subsequently confirm through experimental validation. We optimize deep learning models to decode regulatory grammar, achieving robust modeling of tissue-specific chromatin accessibility. This approach allows to predict cross-variety regulatory dynamics from genomic sequences, shedding light on the genetic underpinnings of cis-regulatory divergence and morphological disparities between varieties. Overall, our study establishes a foundational resource for rice functional genomics and precision molecular breeding, providing valuable insights into regulatory mechanisms governing complex traits.
Asunto(s)
Cromatina , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Estudio de Asociación del Genoma Completo , Oryza , Oryza/genética , Oryza/crecimiento & desarrollo , Cromatina/metabolismo , Cromatina/genética , Mapeo Cromosómico/métodos , Sitios de Carácter Cuantitativo/genética , Germinación/genética , Elementos de Facilitación Genéticos/genética , Aprendizaje Profundo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Although the chloroplast genome (cpDNA) of higher plants is known to exist as a large protein-DNA complex called 'plastid nucleoid', researches on its DNA state and regulatory elements are limited. In this study, we performed the assay for transposase-accessible chromatin sequencing (ATAC-seq) on five common tissues across five grasses, and found that the accessibility of different regions in cpDNA varied widely, with the transcribed regions being highly accessible and accessibility patterns around gene start and end sites varying depending on the level of gene expression. Further analysis identified a total of 3970 putative protein binding footprints on cpDNAs of five grasses. These footprints were enriched in intergenic regions and co-localized with known functional elements. Footprints and their flanking accessibility varied dynamically among tissues. Cross-species analysis showed that footprints in coding regions tended to overlap non-degenerate sites and contain a high proportion of highly conserved sites, indicating that they are subject to evolutionary constraints. Taken together, our results suggest that the accessibility of cpDNA has biological implications and provide new insights into the transcriptional regulation of chloroplasts.
Asunto(s)
Genoma del Cloroplasto , Poaceae , Poaceae/genética , ADN de Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas , Cloroplastos/genética , Cloroplastos/metabolismoRESUMEN
Bermudagrass (Cynodon dactylon) is a globally distributed, extensively used warm-season turf and forage grass with high tolerance to salinity and drought stress in alkaline environments. However, the origin of the species and genetic mechanisms for salinity tolerance in the species are basically unknown. Accordingly, we set out to study evolution divergence events in the Cynodon genome and to identify genes for salinity tolerance. We developed a 604.0 Mb chromosome-level polyploid genome sequence for bermudagrass 'A12359' (n = 18). The C. dactylon genome comprises 2 complete sets of homoeologous chromosomes, each with approximately 30 000 genes, and most genes are conserved as syntenic pairs. Phylogenetic study showed that the initial Cynodon species diverged from Oropetium thomaeum approximately 19.7-25.4 million years ago (Mya), the A and B subgenomes of C. dactylon diverged approximately 6.3-9.1 Mya, and the bermudagrass polyploidization event occurred 1.5 Mya on the African continent. Moreover, we identified 82 candidate genes associated with seven agronomic traits using a genome-wide association study, and three single-nucleotide polymorphisms were strongly associated with three salt resistance genes: RAP2-2, CNG channels, and F14D7.1. These genes may be associated with enhanced bermudagrass salt tolerance. These bermudagrass genomic resources, when integrated, may provide fundamental insights into evolution of diploid and tetraploid genomes and enhance the efficacy of comparative genomics in studying salt tolerance in Cynodon.
Asunto(s)
Cynodon , Genoma de Planta , Filogenia , Tolerancia a la Sal , Secuenciación Completa del Genoma , Cynodon/genética , Tolerancia a la Sal/genética , Genoma de Planta/genética , Tetraploidía , Poliploidía , Cromosomas de las Plantas/genética , Genes de Plantas/genéticaRESUMEN
Panicle architecture is a key determinant of rice grain yield and is mainly determined at the 1-2 mm young panicle stage. Here, we investigated the transcriptome of the 1-2 mm young panicles from 275 rice varieties and identified thousands of genes whose expression levels were associated with panicle traits. Multimodel association studies suggested that many small-effect genetic loci determine spikelet per panicle (SPP) by regulating the expression of genes associated with panicle traits. We found that alleles at cis-expression quantitative trait loci of SPP-associated genes underwent positive selection, with a strong preference for alleles increasing SPP. We further developed a method that integrates the associations of cis- and trans-expression components of genes with traits to identify causal genes at even small-effect loci and construct regulatory networks. We identified 36 putative causal genes of SPP, including SDT (MIR156j) and OsMADS17, and inferred that OsMADS17 regulates SDT expression, which was experimentally validated. Our study reveals the impact of regulatory variants on rice panicle architecture and provides new insights into the gene regulatory networks of panicle traits.
Asunto(s)
Oryza , Transcriptoma , Transcriptoma/genética , Oryza/genética , Oryza/metabolismo , Redes Reguladoras de Genes , Perfilación de la Expresión Génica , Sitios de Carácter Cuantitativo/genéticaRESUMEN
Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypium genus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.
Asunto(s)
Genómica , Gossypium , Gossypium/genética , CromatinaRESUMEN
BACKGROUND: Regulation of gene expression plays an essential role in controlling the phenotypes of plants. Brassica napus (B. napus) is an important source for the vegetable oil in the world, and the seed oil content is an important trait of B. napus. RESULTS: We perform a comprehensive analysis of the transcriptional variability in the seeds of B. napus at two developmental stages, 20 and 40 days after flowering (DAF). We detect 53,759 and 53,550 independent expression quantitative trait loci (eQTLs) for 79,605 and 76,713 expressed genes at 20 and 40 DAF, respectively. Among them, the local eQTLs are mapped to the adjacent genes more frequently. The adjacent gene pairs are regulated by local eQTLs with the same open chromatin state and show a stronger mode of expression piggybacking. Inter-subgenomic analysis indicates that there is a feedback regulation for the homoeologous gene pairs to maintain partial expression dosage. We also identify 141 eQTL hotspots and find that hotspot87-88 co-localizes with a QTL for the seed oil content. To further resolve the regulatory network of this eQTL hotspot, we construct the XGBoost model using 856 RNA-seq datasets and the Basenji model using 59 ATAC-seq datasets. Using these two models, we predict the mechanisms affecting the seed oil content regulated by hotspot87-88 and experimentally validate that the transcription factors, NAC13 and SCL31, positively regulate the seed oil content. CONCLUSIONS: We comprehensively characterize the gene regulatory features in the seeds of B. napus and reveal the gene networks regulating the seed oil content of B. napus.
Asunto(s)
Brassica napus , Brassica napus/genética , Brassica napus/metabolismo , Redes Reguladoras de Genes , Semillas/genética , Semillas/metabolismo , Sitios de Carácter Cuantitativo , Aceites de Plantas/metabolismoRESUMEN
Symbiosis between legumes and rhizobia results in the formation of nitrogen-fixing root nodules. Endoreduplication is essential for nodule development and efficient nitrogen fixation; however, the cellular mechanism by which rhizobial infection causes endoreduplication in symbiotic nodules and the roles of the resulting polyploid cells in nitrogen fixation remain largely unknown. Here, we developed a series of different approaches to separate infected cells (ICs) and uninfected cells (UCs) and determined their ploidy levels in soybean (Glycine max) developing nodules. We demonstrated that 4C nuclei exist in both UCs and ICs of developing nodules and that these 4C cells are primarily invaded by rhizobia and subsequently undergo endoreduplication. Furthermore, RNA-sequencing analysis of nuclei with different ploidy levels from soybean nodules at 12 d post-infection (dpi) and 20 dpi showed that 4C cells are predominantly ICs in 12-dpi nodules but UCs in 20-dpi nodules. We conclude that the infection of 4C cells by rhizobia is critical for initiating endoreduplication. These findings provide significant insight into rhizobial infection, nodule endoreduplication and nitrogen fixation in symbiotic nodules.
Asunto(s)
Fabaceae , Rhizobium , Endorreduplicación , Fijación del Nitrógeno , Nódulos de las Raíces de las Plantas , Glycine max/genética , SimbiosisRESUMEN
Interpreting the functional impacts of genetic variants (GVs) is an important challenge for functional genomic studies in crops and next-generation breeding. Previous studies in rice (Oryza sativa) have focused mainly on the identification of GVs, whereas systematic functional annotation of GVs has not yet been performed. Here, we present a functional impact map of GVs in rice. We curated haplotype information for 17 397 026 GVs from sequencing data of 4726 rice accessions. We quantitatively evaluated the effects of missense mutations in coding regions in each haplotype based on the conservation of amino acid residues and obtained the effects of 918 848 non-redundant missense GVs. Furthermore, we generated high-quality chromatin accessibility (CA) data from six representative rice tissues and used these data to train deep convolutional neural network models to predict the impacts of 5 067 405 GVs for CA in regulatory regions. We characterized the functional properties and tissue specificity of the GV effects and found that large-effect GVs in coding and regulatory regions may be subject to selection in different directions. Finally, we demonstrated how the functional impact map could be used to prioritize causal variants in mapping populations. This impact map will be a useful resource for accelerating gene cloning and functional studies in rice, and can be freely queried in RiceVarMap V2.0 (http://ricevarmap.ncpgr.cn).
Asunto(s)
Bases de Datos de Ácidos Nucleicos , Variación Genética , Genoma de Planta , Oryza/genética , Genotipo , Haplotipos , Mutación INDEL , Polimorfismo de Nucleótido SimpleRESUMEN
Targeting inhibitory immune checkpoint molecules has significantly altered cancer treatment regimens. T cell immunoglobulin and mucin domain 3 (TIM3) is one of the major inhibitory immune checkpoints expressed on T cells. Blocking the engagement of TIM3 and its inhibitory ligand galectin-9 may potentiate the effects of immunotherapy or overcome the adaptive resistance to the therapeutic blockade of programmed cell death protein 1, cytotoxic T-lymphocyte-associated protein 4, B- and T-lymphocyte attenuator and lymphocyte-activation gene 3, amongst others, as each of these immune checkpoints harbors unique properties that set it apart from the rest. Heavy chain variable fragment (VH)-derived single-domain antibodies (sdAbs) represent a class of expanding drug candidates. These sdAbs have unique advantages, including their minimal size in the antibody class, ease of expression, broad scope for modular structure design and re-engineering, and excellent tumor penetration. In the present study, two sdAbs, TIM3-R23 and TIM3-R53, were generated by immunizing rabbits with the recombinant extracellular domain of TIM3 and applying phage display technology. These sdAbs were easily expressed in mammalian cells. The purified sdAbs were able to bind to both recombinant and cell surface TIM3, and blocked it from binding to the ligand galectin-9. In vivo studies demonstrated that TIM3-R53 was able to potentiate the antitumor activity of chimeric antigen receptor T cells that targeted mesothelin. In conclusion, the results of the present study suggested that TIM3-R53 may be a novel and attractive immune checkpoint inhibitor against TIM3, which is worthy of further investigation.
RESUMEN
Reproductive transition of grasses is characterized by switching the pattern of lateral branches, featuring the suppression of outgrowth of the subtending leaves (bracts) and rapid formation of higher-order branches in the inflorescence (panicle). However, the molecular mechanisms underlying such changes remain largely unknown. Here, we show that bract suppression is required for the reproductive branching in rice. We identified a pathway involving the intrinsic time ruler microRNA156/529, their targets SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, NECK LEAF1 (NL1), and PLASTOCHRON1 (PLA1), which regulates the bract outgrowth and thus affects the pattern switch between vegetative and reproductive branching. Suppression of the bract results in global reprogramming of transcriptome and chromatin accessibility following the reproductive transition, while these processes are largely dysregulated in the mutants of these genes. These discoveries contribute to our understanding of the dynamic plant architecture and provide novel insights for improving crop yields.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , MicroARNs/fisiología , Oryza/crecimiento & desarrollo , Proteínas de Plantas/fisiología , ARN de Planta/fisiología , Proteínas de Unión al ARN/fisiología , Factores de Transcripción/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genes de Plantas , Oryza/genética , Desarrollo de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Dedos de ZincRESUMEN
Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high-quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single-molecule real-time sequencing, Hi-C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2-12 was specifically expressed in seeds and was vital for high-LA content in seeds, while tandemly duplicated CarFAD2 genes were up-regulated in ovaries compared to CarFAD2-12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem-duplicated CarCHS5ËCarCHS6, which were up-regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high-quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower.
Asunto(s)
Carthamus tinctorius , Carthamus tinctorius/genética , Cromosomas , Flavonoides , Ácido Linoleico , Semillas/genéticaRESUMEN
ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq .
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Huella de ADN/métodos , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/genética , Arabidopsis/genética , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Secuenciación de Inmunoprecipitación de Cromatina/normas , ADN/análisis , ADN/genética , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
BACKGROUND: Small cell lung cancer (SCLC) accounts for 15% of lung cancers, and the primary treatment of this malignancy is chemotherapy and radiotherapy. Delta-like 3 (DLL3) is an attractive target for SCLC immunotherapy since its expression is highly restricted to SCLC with a neglectable appearance on normal adult tissues. In the current study, we aimed to explore the efficacy of DLL3-targeted SCLC immunotherapy via the engagement of T cell. METHODS: As a proof of concept, we constructed DLL3-targeted bispecific antibody and chimeric antigen receptor (CAR)-modified T cells. In vitro and in vivo tumor-suppression activity of these treatments alone or in combination with a Program Death-1 (PD-1) inhibitory antibody was evaluated. RESULTS: In vitro studies showed that both DLL3 bispecific antibody and CAR-T efficiently killed DLL3-positive cancer cells, including the native SCLC cell lines H446, H196, H82, and the artificial A431 cells that were forcefully overexpressing DLL3. In vivo studies in xenograft mouse models demonstrated that both bispecific antibody and CAR-T suppressed the tumor growth, and combination therapy with PD-1 inhibitory antibody dramatically improved the efficacy of the DLL3 bispecific antibody, but not the CAR-T cells. CONCLUSIONS: Our results demonstrated that DLL3-targeted bispecific antibody plus PD-1 inhibition was effective in controlling SCLC growth.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inmunoterapia Adoptiva/métodos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Neoplasias Pulmonares/terapia , Proteínas de la Membrana/antagonistas & inhibidores , Carcinoma Pulmonar de Células Pequeñas/terapia , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Péptidos y Proteínas de Señalización Intracelular/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Proteínas de la Membrana/inmunología , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Prueba de Estudio Conceptual , Receptores Quiméricos de Antígenos/inmunología , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epidermal growth factor receptor (EGFR) mutation status is the best predictor of patient response to treatments with tyrosine kinase inhibitors in primary lung adenocarcinoma and is typically analyzed by DNA-based techniques, such as direct DNA sequencing and allele-specific PCR. Recently, however, two mutation-specific antibodies against delE746-A750 in exon 19 and L858R in exon 21 have opened the door for a more convenient and more efficient strategy to determine EGFR mutation status. To evaluate the clinical application of a new mutation-specific mouse monoclonal antibody for EGFR (L858R), we performed immunohistochemistry (IHC) studies with tumor samples from primary lung adenocarcinoma in retrospective and validation settings. A total of 215 cases of primary lung adenocarcinoma were examined and compared using a combination of DNA-based techniques (direct DNA sequencing and/or allele-specific PCR) and protein-based IHC. IHC staining was assessed on a 0 to 3+ score scale, and a cutoff value of 2+ was used as positive by IHC. In the retrospective setting, statistical analyses of the data showed that the sensitivity of IHC was 90.9% and the specificity was 96.8%. Findings from the validation study demonstrated that the sensitivity and specificity of IHC were 88.2% and 100%, respectively. IHC with the novel mutation-specific antibody could be used as a screening method to assess the EGFR L858R mutation status in primary lung adenocarcinoma.
Asunto(s)
Adenocarcinoma/genética , Receptores ErbB/biosíntesis , Inmunohistoquímica , Neoplasias Pulmonares/genética , Mutación , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Adulto , Anciano , Animales , Anticuerpos Monoclonales/inmunología , Detección Precoz del Cáncer , Receptores ErbB/genética , Femenino , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patología , Masculino , Ratones , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
With the increased uses of targeted therapeutics, diagnostic detection of target mutations becomes essential for the effective clinical applications of targeted therapeutics. Currently, there are two types of methods detecting target mutations in clinics: one is based on DNA sequence and the other uses the newly developed mutation-specific antibodies recognizing mutated proteins. Each method has its own advantages and disadvantages. Here, we explored the sensitivity and specificity of a new commercially available BRAF(V600E) mutation-specific mouse monoclonal antibody. Using routine manual immunohistochemistry (IHC), we tested tumor tissues from 38 melanoma patients. For those melanoma tissues with abundant endogenous melanin, we pretreated the tumor tissues with 3 % hydrogen peroxide to remove melanin for reliable signal detection. We also performed DNA sequencing and ARMS-PCR analyses for these 38 tumor samples. Comparing to the results from DNA-based detection methods, the IHC method with this BRAF(V600E) mutation-specific antibody displayed 100 % sensitivity and 92.9 % specificity. Hence, this IHC detection is sensitive for clinic uses as a simple, fast, inexpensive, and reliable method to screen cancer patients for the BRAF(V600E) mutation and could be easily adapted for use in most hospital pathology laboratories.
Asunto(s)
Inmunohistoquímica/métodos , Melanoma/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Análisis de Secuencia de ADNRESUMEN
A new, simple and sensitive method for the quantitative analysis of cytochrome C (Cyt C) based on the reduction wave of guanidine modified Co(II)-Cyt C complex at about -1.74 V (vs. SCE) by single sweep polarography in the solution containing 8 x 10(-6) mol L(-1) CoCl2, 0.04 mol L(-1) guanidine hydrochloride, 0.2 mol L(-1) NaOH and 0.5% Na2SO3. The peak height is linearly proportional to the concentration of Cyt C in the range of 0.005 approximately 1.500 mg L(-1) (correlation coefficient 0.999). Common amino acids, saccharide, organic acid and metal ions of appropriate concentrations have no interference on the Cyt C determination. The released Cyt C in the process of mitochondrial permeability transition of Hong-Lian cytoplasmic male sterile line of rice has been measured by the method, and the result is satisfactory.