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To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro-expressed TF, and TF-DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be derived. A researcher with molecular biology experience should be able to follow this protocol, processing up to 400 samples per week.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

Abstract The Plant Cell Atlas (PCA) community hosted a virtual symposium on December 9 and 10, 2021 on single cell and spatial omics technologies. The conference gathered almost 500 academic, industry, and government leaders to identify the needs and directions of the PCA community and to explore how establishing a data synthesis center would address these needs and accelerate progress. This report details the presentations and discussions focused on the possibility of a data synthesis center for a PCA and the expected impacts of such a center on advancing science and technology globally. Community discussions focused on topics such as data analysis tools and annotation standards; computational expertise and cyber‐infrastructure; modes of community organization and engagement; methods for ensuring a broad reach in the PCA community; recruitment, training, and nurturing of new talent; and the overall impact of the PCA initiative. These targeted discussions facilitated dialogue among the participants to gauge whether PCA might be a vehicle for formulating a data synthesis center. The conversations also explored how online tools can be leveraged to help broaden the reach of the PCA (i.e., online contests, virtual networking, and social media stakeholder engagement) and decrease costs of conducting research (e.g., virtual REU opportunities). Major recommendations for the future of the PCA included establishing standards, creating dashboards for easy and intuitive access to data, and engaging with a broad community of stakeholders. The discussions also identified the following as being essential to the PCA's success: identifying homologous cell‐type markers and their biocuration, publishing datasets and computational pipelines, utilizing online tools for communication (such as Slack), and user‐friendly data visualization and data sharing. In conclusion, the development of a data synthesis center will help the PCA community achieve these goals by providing a centralized repository for existing and new data, a platform for sharing tools, and new analytical approaches through collaborative, multidisciplinary efforts. A data synthesis center will help the PCA reach milestones, such as community‐supported data evaluation metrics, accelerating plant research necessary for human and environmental health.

Next-generation sequencing (NGS)-based methods are revolutionizing biology. Their prevalence requires biologists to be increasingly knowledgeable about computational methods to manage the enormous scale of data. As such, early introduction to NGS analysis and conceptual connection to wet-lab experiments is crucial for training young scientists. However, significant challenges impede the introduction of these methods into the undergraduate classroom, including the need for specialized computer programs and knowledge of computer coding. Here, we describe a semester-long, course-based undergraduate research experience at a liberal arts college combining RNA-sequencing (RNA-seq) analysis with student-driven, wet-lab experiments to investigate plant responses to light. Students derived hypotheses based on analysis of RNA-seq data and designed follow-up studies of gene expression and plant growth. Our assessments indicate that students acquired knowledge of big data analysis and computer coding; however, earlier exposure to computational methods may be beneficial. Our course requires minimal prior knowledge of plant biology, is easy to replicate, and can be modified to a shorter, directed-inquiry module. This framework promotes exploration of the links between gene expression and phenotype using examples that are clear and tractable and improves computational skills and bioinformatics self-efficacy to prepare students for the "big data" era of modern biology.

The epigenome orchestrates genome accessibility, functionality, and three-dimensional structure. Because epigenetic variation can impact transcription and thus phenotypes, it may contribute to adaptation. Here, we report 1,107 high-quality single-base resolution methylomes and 1,203 transcriptomes from the 1001 Genomes collection of Arabidopsis thaliana. Although the genetic basis of methylation variation is highly complex, geographic origin is a major predictor of genome-wide DNA methylation levels and of altered gene expression caused by epialleles. Comparison to cistrome and epicistrome datasets identifies associations between transcription factor binding sites, methylation, nucleotide variation, and co-expression modules. Physical maps for nine of the most diverse genomes reveal how transposons and other structural variants shape the epigenome, with dramatic effects on immunity genes. The 1001 Epigenomes Project provides a comprehensive resource for understanding how variation in DNA methylation contributes to molecular and non-molecular phenotypes in natural populations of the most studied model plant.

The complete mitochondrial genome of Fieberiella septentrionalis was annotated for the first time in the present study. The mitogenome was found to have circular shape, with 16,175 bp in size, containing 13 protein coding genes (PCGs), 22 transfer-RNA genes, 2 ribosomal-RNA genes, and 1 non-coding region. The nucleotide composition biases toward A and T is 77.9% of the entirety, which is a typical structure of Cicadellidae. All PCGs have ATN as the start codon, TAA and single T as the stop codon. The resulting phylogenetic tree confirms that the F. septentrionalis belongs to the subfamily of Deltocephalinae and Fieberiellini as sister to the remaining tribes of this subfamily.

Gene regulatory networks (GRNs) drive organism structure and functions, so the discovery and characterization of GRNs is a major goal in biological research. However, accurate identification of causal regulatory connections and inference of GRNs using gene expression datasets, more recently from single-cell RNA-seq (scRNA-seq), has been challenging. Here we employ the innovative method of Causal Inference Using Composition of Transactions (CICT) to uncover GRNs from scRNA-seq data. The basis of CICT is that if all gene expressions were random, a non-random regulatory gene should induce its targets at levels different from the background random process, resulting in distinct patterns in the whole relevance network of gene–gene associations. CICT proposes novel network features derived from a relevance network, which enable any machine learning algorithm to predict causal regulatory edges and infer GRNs. We evaluated CICT using simulated and experimental scRNA-seq data in a well-established benchmarking pipeline and showed that CICT outperformed existing network inference methods representing diverse approaches with many-fold higher accuracy. Furthermore, we demonstrated that GRN inference with CICT was robust to different levels of sparsity in scRNA-seq data, the characteristics of data and ground truth, the choice of association measure and the complexity of the supervised machine learning algorithm. Our results suggest aiming at directly predicting causality to recover regulatory relationships in complex biological networks substantially improves accuracy in GRN inference. [ABSTRACT FROM AUTHOR]

Icariin has been demonstrated to stimulate mesenchymal stem cell (MSC) osteogensis and activate several signals, such as PI3K/Akt, but how the osteogenesis was sequentially mediated is unclear. Runx2 is one of the osteogenic regulators in MSC and is regulated by the TAZ gene. The purpose of this study was to investigate whether icariin-activated PI3K/Akt crosstalked with the TAZ-Runx2 pathway to regulate MSC osteogenesis. Adipose-derived MSCs were treated with icariin alone, together with TAZ silencing or PI3K/Akt inhibitor. Normal MSCs were used as a control. The activation of PI3K/Akt, expression of TAZ and downstream expression of Runx2 were analyzed. Induction of MSC osteogenesis under different treatments was detected. The results demonstrated that icariin treatment significantly activated PI3K/Akt and TAZ expression, as well as the downstream Runx2 expression. When activation of PI3K/Akt by icariin was inhibited by LY294002, upregulated TAZ expression was reversed, as well as the downstream expression of Runx2. Consequently, with the osteogenic counteracting effects of icariin on MSCs, inhibition of TAZ upregulation by siRNA did not significantly influence PI3K/ Akt activation in icariin-treated MSCs, but icariin-induced upregulation of Runx2 and osteogenic differentiation in MSCs was counteracted. It could be concluded from these findings that icariin treatment activated PI3K/Akt and further mediated the transcriptional activation of the TAZ/Runx2 pathway to induce osteogenic differentiation of MSCs.