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Nitrogen (N) and Water (W) - two resources critical for crop productivity – are becoming increasingly limited in soils globally. To address this issue, we aim to uncover the gene regulatory networks (GRNs) that regulate nitrogen use efficiency (NUE) - as a function of water availability - in Oryza sativa, a staple for 3.5 billion people. In this study, we infer and validate GRNs that correlate with rice NUE phenotypes affected by N-by-W availability in the field. We did this by exploiting RNA-seq and crop phenotype data from 19 rice varieties grown in a 2x2 N-by-W matrix in the field. First, to identify gene-to-NUE field phenotypes, we analyzed these datasets using weighted gene co-expression network analysis (WGCNA). This identified two network modules ("skyblue" & "grey60") highly correlated with NUE grain yield (NUEg). Next, we focused on 90 TFs contained in these two NUEg modules and predicted their genome-wide targets using the N-and/or-W response datasets using a random forest network inference approach (GENIE3). Next, to validate the GENIE3 TF→target gene predictions, we performed Precision/Recall Analysis (AUPR) using nine datasets for three TFs validated in planta . This analysis sets a precision threshold of 0.31, used to "prune" the GENIE3 network for high-confidence TF→target gene edges, comprising 88 TFs and 5,716 N-and/or-W response genes. Next, we ranked these 88 TFs based on their significant influence on NUEg target genes responsive to N and/or W signaling. This resulted in a list of 18 prioritized TFs that regulate 551 NUEg target genes responsive to N and/or W signals. We validated the direct regulated targets of two of these candidate NUEg TFs in a plant cell-based TF assay called TARGET, for which we also had in planta data for comparison. Gene ontology analysis revealed that 6/18 NUEg TFs - OsbZIP23 (LOC_Os02g52780), Oshox22 (LOC_Os04g45810), LOB39 (LOC_Os03g41330), Oshox13 (LOC_Os03g08960), LOC_Os11g38870, and LOC_Os06g14670 - regulate genes annotated for N and/or W signaling. Our results show that OsbZIP23 and Oshox22, known regulators of drought tolerance, also coordinate W-responses with NUEg. This validated network can aid in developing/breeding rice with improved yield on marginal, low N-input, drought-prone soils.
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ConnecTF: A platform to integrate transcription factor–gene interactions and validate regulatory networks
Abstract Deciphering gene regulatory networks (GRNs) is both a promise and challenge of systems biology. The promise lies in identifying key transcription factors (TFs) that enable an organism to react to changes in its environment. The challenge lies in validating GRNs that involve hundreds of TFs with hundreds of thousands of interactions with their genome-wide targets experimentally determined by high-throughput sequencing. To address this challenge, we developed ConnecTF, a species-independent, web-based platform that integrates genome-wide studies of TF–target binding, TF–target regulation, and other TF-centric omic datasets and uses these to build and refine validated or inferred GRNs. We demonstrate the functionality of ConnecTF by showing how integration within and across TF–target datasets uncovers biological insights. Case study 1 uses integration of TF–target gene regulation and binding datasets to uncover TF mode-of-action and identify potential TF partners for 14 TFs in abscisic acid signaling. Case study 2 demonstrates how genome-wide TF–target data and automated functions in ConnecTF are used in precision/recall analysis and pruning of an inferred GRN for nitrogen signaling. Case study 3 uses ConnecTF to chart a network path from NLP7, a master TF in nitrogen signaling, to direct secondary TF2s and to its indirect targets in a Network Walking approach. The public version of ConnecTF (https://ConnecTF.org) contains 3,738,278 TF–target interactions for 423 TFs in Arabidopsis, 839,210 TF–target interactions for 139 TFs in maize (Zea mays), and 293,094 TF–target interactions for 26 TFs in rice (Oryza sativa). The database and tools in ConnecTF will advance the exploration of GRNs in plant systems biology applications for model and crop species.
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- Award ID(s):
- 1840761
- PAR ID:
- 10231627
- Date Published:
- Journal Name:
- Plant Physiology
- Volume:
- 185
- Issue:
- 1
- ISSN:
- 0032-0889
- Page Range / eLocation ID:
- 49 to 66
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The regulation of gene expression is central to many biological processes. Gene regulatory networks (GRNs) link transcription factors (TFs) to their target genes and represent maps of potential transcriptional regulation. Here, we analyzed a large number of publically available maize (Zea mays) transcriptome data sets including >6000 RNA sequencing samples to generate 45 coexpression-based GRNs that represent potential regulatory relationships between TFs and other genes in different populations of samples (cross-tissue, cross-genotype, and tissue-and-genotype samples). While these networks are all enriched for biologically relevant interactions, different networks capture distinct TF-target associations and biological processes. By examining the power of our coexpression-based GRNs to accurately predict covarying TF-target relationships in natural variation data sets, we found that presence/absence changes rather than quantitative changes in TF gene expression are more likely associated with changes in target gene expression. Integrating information from our TF-target predictions and previous expression quantitative trait loci (eQTL) mapping results provided support for 68 TFs underlying 74 previously identified trans-eQTL hotspots spanning a variety of metabolic pathways. This study highlights the utility of developing multiple GRNs within a species to detect putative regulators of important plant pathways and provides potential targets for breeding or biotechnological applications.more » « less
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Marshall-Colon, Amy (Ed.)Abstract Gene regulatory networks (GRNs) are defined by a cascade of transcriptional events by which signals, such as hormones or environmental cues, change development. To understand these networks, it is necessary to link specific transcription factors (TFs) to the downstream gene targets whose expression they regulate. Although multiple methods provide information on the targets of a single TF, moving from groups of co-expressed genes to the TF that controls them is more difficult. To facilitate this bottom-up approach, we have developed a web application named TF DEACoN. This application uses a publicly available Arabidopsis thaliana DNA Affinity Purification (DAP-Seq) data set to search for TFs that show enriched binding to groups of co-regulated genes. We used TF DEACoN to examine groups of transcripts regulated by treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), using a transcriptional data set performed with high temporal resolution. We demonstrate the utility of this application when co-regulated genes are divided by timing of response or cell-type-specific information, which provides more information on TF/target relationships than when less defined and larger groups of co-regulated genes are used. This approach predicted TFs that may participate in ethylene-modulated root development including the TF NAM (NO APICAL MERISTEM). We used a genetic approach to show that a mutation in NAM reduces the negative regulation of lateral root development by ACC. The combination of filtering and TF DEACoN used here can be applied to any group of co-regulated genes to predict GRNs that control coordinated transcriptional responses.more » « less
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null (Ed.)All aspects of transcription and its regulation involve dynamic events. However, capturing these dynamic events in gene regulatory networks (GRNs) offers both a promise and a challenge. The promise is that capturing and modeling the dynamic changes in GRNs will allow us to understand how organisms adapt to a changing environment. The ability to mount a rapid transcriptional response to environmental changes is especially important in nonmotile organisms such as plants. The challenge is to capture these dynamic, genome-wide events and model them in GRNs. In this review, we cover recent progress in capturing dynamic interactions of transcription factors with their targets—at both the local and genome-wide levels—and using them to learn how GRNs operate as a function of time. We also discuss recent advances that employ time-based machine learning approaches to forecast gene expression at future time points, a key goal of systems biology. Expected final online publication date for the Annual Review of Plant Biology, Volume 72 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.more » « less
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Understanding the dynamics of gene regulatory networks (GRNs) across diverse cell types poses a challenge yet holds immense value in unraveling the molecular mechanisms governing cellular processes. Current computational methods, which rely solely on expression changes from bulk RNA-seq and/or scRNA-seq data, often result in high rates of false positives and low precision. Here, we introduce an advanced computational tool, DeepIMAGER, for inferring cell-specific GRNs through deep learning and data integration. DeepIMAGER employs a supervised approach that transforms the co-expression patterns of gene pairs into image-like representations and leverages transcription factor (TF) binding information for model training. It is trained using comprehensive datasets that encompass scRNA-seq profiles and ChIP-seq data, capturing TF-gene pair information across various cell types. Comprehensive validations on six cell lines show DeepIMAGER exhibits superior performance in ten popular GRN inference tools and has remarkable robustness against dropout-zero events. DeepIMAGER was applied to scRNA-seq datasets of multiple myeloma (MM) and detected potential GRNs for TFs of RORC, MITF, and FOXD2 in MM dendritic cells. This technical innovation, combined with its capability to accurately decode GRNs from scRNA-seq, establishes DeepIMAGER as a valuable tool for unraveling complex regulatory networks in various cell types.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/plphys/kiaa012
