Transcription factor binding site divergence across maize inbred lines drives transcriptional and phenotypic variation.

Regulatory elements are important constituents of plant genomes that have shaped ancient and modern crops. Their identification, function, and diversity in crop genomes however are poorly characterized, thus limiting our ability to harness their power for further agricultural advances using induced or natural variation. Here, we use DNA affinity purification-sequencing (DAP-seq) to map transcription factor (TF) binding events for 200 maize TFs belonging to 30 distinct families and heterodimer pairs in two distinct inbred lines historically used for maize hybrid plant production, providing empirical binding site annotation for 5.3% of the maize genome. TF binding site comparison in B73 and Mo17 inbreds reveals widespread differences, driven largely by structural variation, that correlate with gene expression changes. TF binding site presence-absence variation helps clarify complex QTL such as vgt1, an important determinant of maize flowering time, and DICE, a distal enhancer involved in herbivore resistance. Modification of TF binding regions via CRISPR-Cas9 mediated editing alters target gene expression and phenotype. Our functional catalog of maize TF binding events enables collective and comparative TF binding analysis, and highlights its value for agricultural improvement.

Robust discovery of gene regulatory networks from single-cell gene expression data by Causal Inference Using Composition of Transactions.

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.

MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors.

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo- versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4 cis-elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.

Transcription Factor Dynamics in Cross-Regulation of Plant Hormone Signaling Pathways.

Cross-regulation between hormone signaling pathways is indispensable for plant growth and development. However, the molecular mechanisms by which multiple hormones interact and co-ordinate activity need to be understood. Here, we generated a cross-regulation network explaining how hormone signals are integrated from multiple pathways in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. To do so we comprehensively characterized transcription factor activity during plant hormone responses and reconstructed dynamic transcriptional regulatory models for six hormones; abscisic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and strigolactone/karrikin. These models incorporated target data for hundreds of transcription factors and thousands of protein-protein interactions. Each hormone recruited different combinations of transcription factors, a subset of which were shared between hormones. Hub target genes existed within hormone transcriptional networks, exhibiting transcription factor activity themselves. In addition, a group of MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) were identified as potential key points of cross-regulation between multiple hormones. Accordingly, the loss of function of one of these (MPK6) disrupted the global proteome, phosphoproteome and transcriptome during hormone responses. Lastly, we determined that all hormones drive substantial alternative splicing that has distinct effects on the transcriptome compared with differential gene expression, acting in early hormone responses. These results provide a comprehensive understanding of the common features of plant transcriptional regulatory pathways and how cross-regulation between hormones acts upon gene expression.

Kartogenin (KGN)/synthetic melanin nanoparticles (SMNP) loaded theranostic hydrogel scaffold system for multiparametric magnetic resonance imaging guided cartilage regeneration.

Cartilage regeneration after injury is still a great challenge in clinics, which suffers from its avascularity and poor proliferative ability. Herein we designed a novel biocompatible cellulose nanocrystal/GelMA (gelatin-methacrylate anhydride)/HAMA (hyaluronic acid-methacrylate anhydride)-blended hydrogel scaffold system, loaded with synthetic melanin nanoparticles (SMNP) and a bioactive drug kartogenin (KGN) for theranostic purpose. We found that the SMNP-KGN/Gel showed favorable mechanical property, thermal stability, and distinct magnetic resonance imaging (MRI) contrast enhancement. Meanwhile, the sustained release of KGN could recruit bone-derived mesenchymal stem cells to proliferate and differentiate into chondrocytes, which promoted cartilage regeneration in vitro and in vivo. The hydrogel degradation and cartilage restoration were simultaneously monitored by multiparametric MRI for 12 weeks, and further confirmed by histological analysis. Together, these results validated the multifunctional hydrogel as a promising tissue engineering platform for noninvasive imaging-guided precision therapy in cartilage regenerative medicine.

First Plant Cell Atlas symposium report.

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.

Elucidating the biology of transcription factor-DNA interaction for accurate identification of cis-regulatory elements.

Transcription factors (TFs) play a critical role in determining cell fate decisions by integrating developmental and environmental signals through binding to specific cis-regulatory modules and regulating spatio-temporal specificity of gene expression patterns. Precise identification of functional TF binding sites in time and space not only will revolutionize our understanding of regulatory networks governing cell fate decisions but is also instrumental to uncover how genetic variations cause morphological diversity or disease. In this review, we discuss recent advances in mapping TF binding sites and characterizing the various parameters underlying the complexity of binding site recognition by TFs.

Direct regulation of shikimate, early phenylpropanoid, and stilbenoid pathways by Subgroup 2 R2R3-MYBs in grapevine.

The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis vinifera L.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we show how DNA affinity purification sequencing (DAP-Seq) allows for the genome-wide TF-binding site interrogation in grape. We obtained 5190 and 4443 binding events assigned to 4041 and 3626 genes for MYB14 and MYB15, respectively (approximately 40% of peaks located within −10 kb of transcription start sites). DAP-Seq of MYB14/MYB15 was combined with aggregate gene co-expression networks (GCNs) built from more than 1400 transcriptomic datasets from leaves, fruits, and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15, and MYB13, a third uncharacterized member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 of 47 STS family genes. Moreover, all three MYBs bind to several PAL, C4H, and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene candidates among which ROMT2-3 were validated enzymatically. A high proportion of DAP-Seq bound genes were induced in the activated transcriptomes of transient MYB15-overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3-MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP-Seq and reciprocal GCNs offers a rapid framework for gene function characterization using genome-wide approaches in the context of non-model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialized metabolism.

Vision, challenges and opportunities for a Plant Cell Atlas.

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.

Potentials of single-cell genomics in deciphering cellular phenotypes.

Single-cell genomics, particularly single-cell transcriptome profiling by RNA sequencing have transformed the possibilities to relate genes to functions, structures, and eventually phenotypes. We can now observe changes in each cell's transcriptome and among its neighborhoods, interrogate the sequence of transcriptional events, and assess their influence on subsequent events. This paradigm shift in biology enables us to infer causal relationships in these events with high accuracy. Here we review the latest single-cell studies in plants that uncover how cellular phenotypes emerge as a result of the transcriptome process such as waves of expression, trajectories of development and responses to the environment, and spatial information. With an eye on the advances made in animal and human studies, we further highlight some of the needed areas for future research and development, including computational methods.

Injectable supramolecular gelatin hydrogel loading of resveratrol and histatin-1 for burn wound therapy.

Prolonged inflammatory response and insufficient vascularization cause delayed and poor wound healing. In this study, we fabricated a supramolecular host-guest gelatin (HGM) hydrogel loaded with resveratrol (Res) and histatin-1 (His-1) to suppress inflammation and promote vascularization at skin burn wound sites. The HGM hydrogel showed good properties of shear-thinning and injectability, thereby allowing easy in situ injection and fast adaption to irregular wounds. Res and His-1 were demonstrated to enhance angiogenesis in vitro using cell migration and tube formation assays based on human umbilical vein endothelial cells (HUVECs). In an established rat burn wound model, HGM/Res/His-1 hydrogel treatment promoted wound healing by inhibiting expression of the pro-inflammatory factors of interleukin 6 (IL-6), interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) and increasing the expression of transforming growth factor ß1 (TGF-ß1) and platelet endothelial cell adhesion molecule-1 (CD31). HGM/Res/His-1 hydrogel treatment showed comparable efficacy with that of the commercial dressing, Tegaderm™, and therefore shows promising potential for clinical translation.

Dextran methacrylate hydrogel microneedles loaded with doxorubicin and trametinib for continuous transdermal administration of melanoma.

Microneedles (MNs) technology has many advantages and is an ideal local transdermal drug delivery method. Here we synthesized photocrosslinkable dextran methacrylate (DexMA), and its degree of substitution is 5 % higher than the previous method. We used DexMA hydrogel for the first time to develop a new type of MNs for continuous transdermal administration. The prepared hydrogel MNs can successfully penetrate the epidermal layer and achieve sustained drug release. Doxorubicin (DOX) and trametinib (Tra) are anticancer drugs approved by FDA. Besides, Tra can also reverse P-gp-mediated multidrug resistance (MDR) to effectively block the efflux of DOX by P-gp. We used MNs to simultaneously load Tra and DOX, and achieved synergy in a B16 cell xenograft nude mouse model. The DexMA hydrogel MNs developed in this study can be used to enhance the transdermal delivery of small molecule drugs and reduce systemic toxicity and side effects.

Functionalized GO Nanovehicles with Nitric Oxide Release and Photothermal Activity-Based Hydrogels for Bacteria-Infected Wound Healing.

Bacteria-infected wounds are attracting increasing attention, as antibiotic misuse and multidrug-resistant bacteria complicate their treatment. Herein, we reported a photothermal activity-based drug consisting of ß-cyclodextrin (ßCD)-functionalized graphene oxide (GO) near-infrared light-responsive nanovehicles combined with the nitric oxide donor BNN6, in a methacrylate-modified gelatin (GelMA)/hyaluronic acid graft dopamine (HA-DA) hydrogel. The synergistic effects of photothermal and gas therapies are expected to improve antibacterial efficiency and reduce drug resistance. The results revealed that GelMA/HA-DA/GO-ßCD-BNN6 was an ideal antibacterial material that improved collagen deposition and angiogenesis and promoted wound healing in a mouse model of full-thickness skin repair, compared to the commercially available Aquacel Ag dressing. We developed a multifunctional nanocomposite hydrogel that exhibited antibacterial and angiogenic properties, adhesiveness, and mechanical properties that enhance the regeneration of bacteria-infected wounds.

Cannabinoid exposure in rat adolescence reprograms the initial behavioral, molecular, and epigenetic response to cocaine.

The initial response to an addictive substance can facilitate repeated use: That is, individuals experiencing more positive effects are more likely to use that drug again. Increasing evidence suggests that psychoactive cannabinoid use in adolescence enhances the behavioral effects of cocaine. However, despite the behavioral data, there is no neurobiological evidence demonstrating that cannabinoids can also alter the brain's initial molecular and epigenetic response to cocaine. Here, we utilized a multiomics approach (epigenomics, transcriptomics, proteomics, and phosphoproteomics) to characterize how the rat brain responds to its first encounter with cocaine, with or without preexposure to the synthetic cannabinoid WIN 55,212-2 (WIN). We find that in adolescent (but not in adult) rats, preexposure to WIN results in cross-sensitization to cocaine, which correlates with histone hyperacetylation and decreased levels of HDAC6 in the prefrontal cortex (PFC). In the PFC, we also find that WIN preexposure blunts the typical mRNA response to cocaine and instead results in alternative splicing and chromatin accessibility events, involving genes such as Npas2 Moreover, preexposure to WIN enhances the effects of cocaine on protein phosphorylation, including ERK/MAPK-targets like gephyrin, and modulates the synaptic AMPAR/GluR composition both in the PFC and the nucleus accumbens (NAcc). PFC-NAcc gene network topological analyses, following cocaine exposure, reveal distinct top nodes in the WIN preexposed group, which include PACAP/ADCYAP1. These preclinical data demonstrate that adolescent cannabinoid exposure reprograms the initial behavioral, molecular, and epigenetic response to cocaine.

Big Data to the Bench: Transcriptome Analysis for Undergraduates.

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.

Preparation and characterisation of a novel silk fibroin/hyaluronic acid/sodium alginate scaffold for skin repair.

To mimic the natural structure of tissue extracellular matrix, a novel silk fibroin (SF)/hyaluronic acid (HA)/sodium alginate (SA) composite scaffold (92% in porosity) was prepared by freeze-drying. Fourier-transform infrared spectroscopy and Raman spectra indicated interactions among SF, HA, and SA molecules. Scanning electron microscopy showed that the prepared SF/HA/SA scaffold had soft, elastic characteristics, with an average pore diameter of 93 µm. Mechanical property, thermogravimetric analyses and degradation results indicated that the SF/HA/SA scaffold had good physical stability in body fluid and mechanical movement-related environments. Cell proliferation, morphological, and live-dead analyses showed that NIH-3T3 fibroblast cells were better able to attach, grow, and proliferate on the SF/HA/SA scaffold compared with SF, SF/HA, and SF/SA scaffolds. We evaluated the wound healing effects in a rat full-thickness burn model. The hematoxylin-eosin (H&E) and Masson's trichrome staining results from SF/HA/SA scaffold showed that improved re-epithelialization, enhanced extracellular matrix remodeling. Our findings showed that the prepared SF/HA/SA scaffold can provide a potential way as a wound dressing for skin repair.

Piecing together cis-regulatory networks: insights from epigenomics studies in plants.

5-Methylcytosine, a chemical modification of DNA, is a covalent modification found in the genomes of both plants and animals. Epigenetic inheritance of phenotypes mediated by DNA methylation is well established in plants. Most of the known mechanisms of establishing, maintaining and modifying DNA methylation have been worked out in the reference plant Arabidopsis thaliana. Major functions of DNA methylation in plants include regulation of gene expression and silencing of transposable elements (TEs) and repetitive sequences, both of which have parallels in mammalian biology, involve interaction with the transcriptional machinery, and may have profound effects on the regulatory networks in the cell. Methylome and transcriptome dynamics have been investigated in development and environmental responses in Arabidopsis and agriculturally and ecologically important plants, revealing the interdependent relationship among genomic context, methylation patterns, and expression of TE and protein coding genes. Analyses of methylome variation among plant natural populations and species have begun to quantify the extent of genetic control of methylome variation vs. true epimutation, and model the evolutionary forces driving methylome evolution in both short and long time scales. The ability of DNA methylation to positively or negatively modulate binding affinity of transcription factors (TFs) provides a natural link from genome sequence and methylation changes to transcription. Technologies that allow systematic determination of methylation sensitivities of TFs, in native genomic and methylation context without confounding factors such as histone modifications, will provide baseline datasets for building cell-type- and individual-specific regulatory networks that underlie the establishment and inheritance of complex traits. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Biological Mechanisms > Regulatory Biology.