ETS1, a Target Gene of the EWSR1::FLI1 Fusion Oncoprotein, Regulates the Expression of the Focal Adhesion Protein TENSIN3.

The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma cells reflects the regulatory state of genes associated with the DNA-binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in Ewing sarcoma cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3-repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of Ewing sarcoma cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. Ewing sarcoma cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared with control cells. Visualization of control Ewing sarcoma cells showed a distributed vinculin signal and a network-like organization of F-actin; in contrast, ETS1-activated Ewing sarcoma cells showed an accumulation of vinculin and F-actin toward the plasma membrane. Interestingly, the phenotype of ETS1-activated Ewing sarcoma cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in Ewing sarcoma tumors positively correlates with that of ETS1. Implications: ETS1's transcriptional regulation of the gene encoding the focal adhesion protein TENSIN3 in Ewing sarcoma cells promotes cell movement, a critical step in the evolution of metastasis.

Staufen1 Represses the FOXA1-Regulated Transcriptome by Destabilizing FOXA1 mRNA in Colorectal Cancer Cells.

Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3.

The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma (EWS) cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in EWS cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of EWS cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. EWS cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared to control cells. The cytoskeleton of control cells and ETS1-activated EWS cell lines also differed. Specifically, control cells exhibited a distributed vinculin signal and a network-like organization of F-actin. In contrast, ETS1-activated EWS cells showed an accumulation of vinculin and F-actin towards the plasma membrane. Interestingly, the phenotype of ETS1-activated EWS cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in EWS tumors positively correlates with that of ETS1.

Iron-Sulfur Cluster Protein NITROGEN FIXATION S-LIKE1 and Its Interactor FRATAXIN Function in Plant Immunity.

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are present in all kingdoms of life as part of a large number of proteins involved in several cellular processes, including DNA replication and metabolism. In this work, we demonstrate an additional role for two Fe-S cluster genes in biotic stress responses in plants. Eleven Fe-S cluster genes, including the NITROGEN FIXATION S-LIKE1 (NFS1) and its interactor FRATAXIN (FH), when silenced in Nicotiana benthamiana, compromised nonhost resistance to Pseudomonas syringae pv. tomato T1. NbNFS1 expression was induced by pathogens and salicylic acid. Arabidopsis (Arabidopsis thaliana) atnfs and atfh mutants, with reduced AtNFS1 or AtFH gene expression, respectively, showed increased susceptibility to both host and nonhost pathogen infection. Arabidopsis AtNFS1 and AtFH overexpressor lines displayed decreased susceptibility to infection by host pathogen P syringae pv. tomato DC3000. The AtNFS1 overexpression line exhibited constitutive upregulation of several defense-related genes and enrichment of gene ontology terms related to immunity and salicylic acid responses. Our results demonstrate that NFS1 and its interactor FH are involved not only in nonhost resistance but also in basal resistance, suggesting a new role of the Fe-S cluster pathway in plant immunity.

pssRNAit: A Web Server for Designing Effective and Specific Plant siRNAs with Genome-Wide Off-Target Assessment.

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit The pssRNAit server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element.

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses.

MtSSPdb: The Medicago truncatula Small Secreted Peptide Database.

A growing number of small secreted peptides (SSPs) in plants are recognized as important regulatory molecules with roles in processes such as growth, development, reproduction, stress tolerance, and pathogen defense. Recent discoveries further implicate SSPs in regulating root nodule development, which is of particular significance for legumes. SSP-coding genes are frequently overlooked, because genome annotation pipelines generally ignore small open reading frames, which are those most likely to encode SSPs. Also, SSP-coding small open reading frames are often expressed at low levels or only under specific conditions, and thus are underrepresented in non-tissue-targeted or non-condition-optimized RNA-sequencing projects. We previously identified 4,439 SSP-encoding genes in the model legume Medicago truncatula To support systematic characterization and annotation of these putative SSP-encoding genes, we developed the M. truncatula Small Secreted Peptide Database (MtSSPdb; https://mtsspdb.noble.org/). MtSSPdb currently hosts (1) a compendium of M. truncatula SSP candidates with putative function and family annotations; (2) a large-scale M. truncatula RNA-sequencing-based gene expression atlas integrated with various analytical tools, including differential expression, coexpression, and pathway enrichment analyses; (3) an online plant SSP prediction tool capable of analyzing protein sequences at the genome scale using the same protocol as for the identification of SSP genes; and (4) information about a library of synthetic peptides and root and nodule phenotyping data from synthetic peptide screens in planta. These datasets and analytical tools make MtSSPdb a unique and valuable resource for the plant research community. MtSSPdb also has the potential to become the most complete database of SSPs in plants.

Identification and Functional Investigation of Genome-Encoded, Small, Secreted Peptides in Plants.

Hundreds to thousands of small secreted peptides (SSPs) are encoded in plant genomes but have been overlooked, and most remain unannotated and unstudied. Despite their low profile, they have been found to confer dramatic effects on growth and development of plants. With the growing appreciation of their significance, the development of appropriate methods to identify and functionally assess the myriad SSPs encoded in plant genomes has become critical. Here, we provide protocols for the computational and physiological analysis of SSPs in plant genomes. We first describe our methodology successfully used for genome-wide identification and annotation of SSP-coding genes in the model legume Medicago truncatula, which can be readily adapted for other plant species. We then provide protocols for the functional analysis of SSPs using various synthetic peptide screens. Considerations for the design and handling of peptides are included. © 2019 by John Wiley & Sons, Inc.

Global transcriptome sequencing using the Illumina platform and the development of EST-SSR markers in autotetraploid alfalfa.

BACKGROUND: Alfalfa is the most widely cultivated forage legume and one of the most economically valuable crops in the world. The large size and complexity of the alfalfa genome has delayed the development of genomic resources for alfalfa research. Second-generation Illumina transcriptome sequencing is an efficient method for generating a global transcriptome sequence dataset for gene discovery and molecular marker development in alfalfa. METHODOLOGY/PRINCIPAL FINDINGS: More than 28 million sequencing reads (5.64 Gb of clean nucleotides) were generated by Illumina paired-end sequencing from 15 different alfalfa tissue samples. In total, 40,433 unigenes with an average length of 803 bp were obtained by de novo assembly. Based on a sequence similarity search of known proteins, a total of 36,684 (90.73%) unigenes were annotated. In addition, 1,649 potential EST-SSRs were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 100 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 82 were able to amplify sequences in initial screening tests, and 27 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 10 alfalfa accessions. CONCLUSIONS/SIGNIFICANCE: The present study provided global sequence data for autotetraploid alfalfa and demonstrates the Illumina platform is a fast and effective approach to EST-SSR markers development in alfalfa. The use of these transcriptome datasets will serve as a valuable public information platform to accelerate studies of the alfalfa genome.

A legume specific protein database (LegProt) improves the number of identified peptides, confidence scores and overall protein identification success rates for legume proteomics.

A legume specific protein database (LegProt) has been created containing sequences from seven legume species, i.e., Glycine max, Lotus japonicus, Medicago sativa, Medicago truncatula, Lupinusalbus, Phaseolus vulgaris, and Pisum sativum. The database consists of amino acid sequences translated from predicted gene models and 6-frame translations of tentative consensus (TC) sequences assembled from expressed sequence tags (ESTs) and singleton ESTs. This database was queried using mass spectral data for protein identification and identification success rates were compared to the NCBI nr database. Specifically, Mascot MS/MS ion searches of tandem nano-LC Q-TOFMS/MS mass spectral data showed that relative to the NCBI nr protein database, the LegProt database yielded a 54% increase in the average protein score (i.e., from NCBI nr 480 to LegProt 739) and a 50% increase in the average number of matched peptides (i.e., from NCBI nr 8 to LegProt 12). The overall identification success rate also increased from 88% (NCBI nr) to 93% (LegProt). Mascot peptide mass fingerprinting (PMF) searches of the LegProt database using MALDI-TOFMS data yielded a significant increase in the identification success rate from 19% (NCBI nr) to 34% (LegProt) while the average scores and average number of matched peptides showed insignificant changes. The results demonstrate that the LegProt database significantly increases legume protein identification success rates and the confidence levels compared to the commonly used NCBI nr. These improvements are primarily due to the presence of a large number of legume specific TC sequences in the LegProt database that were not found in NCBI nr. The LegProt database is freely available for download (http://bioinfo.noble.org/manuscript-support/legumedb) and will serve as a valuable resource for legume proteomics.

Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula.

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190,000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R(1) lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14,000-16,000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500,000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.