Putative Gene Regulatory Networks Research Articles

Gene Regulatory Networks Controlling Sinusoidal Hematopoietic Niche Function

The bone marrow sinusoidal vascular niche is important for HSC engraftment and regeneration of hematopoiesis after transplantation, but the epigenetic factors that control these biological functions are undefined. Here, we show that sinusoidal hematopoietic niche functions are controlled by gene regulatory networks (GRNs), involving transcription factors (TFs) that bind to enhancers to regulate corresponding target genes. To characterize sinusoidal niche specific transcriptional and chromatin accessibility states, we performed multimodal single nuclei RNA and ATAC-seq analysis on unsorted zebrafish embryos. We identified 4 niche clusters, including mesenchymal stem cells, fibroblasts, osteoblasts and sinusoidal endothelial cells. Differential RNA transcriptome analysis confirmed the identity of each niche cluster with canonical gene markers for each niche cluster. To examine differential transcriptional accessibility in zebrafish niche clusters, we aggregated co-accessible regions in cells from these clusters. Each cluster demonstrated a unique chromatin accessibility profile with differentially accessible regions. Differential motif activity based on chromatin accessibility analysis further exhibited unique TF motifs activity in sinusoidal endothelial cells. To identify potential regulatory TFs in sinusoidal vascular niche, we predicted TFs based on the TF binding sites accessibility and TF expression. Thus, we identified 3 specific expressed and enriched TFs for sinusoidal vascular niche, including Fli1 (Friend leukemia integration 1), Mef2a (Myocyte enhancer factor 2A) and Mef2c. Next, to investigate genomic regulatory regions that interact with TFs, in which control the transcription of their target genes, we performed random forests and regression trees machine learning methods to infer GRNs. We observed multiple active GRNs in sinusoidal vascular niche, which were predicted to essential for regulating hematopoietic niche development and HSC maintenance, including klf12, egfl7, ebf3 and ldb2. Finally, we examined whether these putative gene regulatory networks were conserved across species and tissue types. We used same approach (single-cell multiome sequencing) on E4-HUVEC cells, a transduced human umbilical vein endothelial cells (HUVEC) with the adenovirus E4ORF1 gene, which was a well-defined model of the human sinusoidal vascular niche. We likewise observed regulatory networks in HUVEC cells. As in zebrafish, GRNs in human sinusoidal cluster consisted of FLI1 and MEF2 family, regulatory regions and their target genes. Our findings reveal that unique sinusoidal vascular niche specific gene regulatory networks, including TFs, regulatory regions and target genes indicate the treatments targeting GRNs may help to facilitate reprogramming of sinusoidal endothelial niche to support HSC homing and regeneration after transplantation.

Deciphering the decisive factors driving fate bifurcations in somatic cell reprogramming

Single-cell studies have demonstrated that somatic cell reprogramming is a continuous process of cell fates transition. Only partial reprogramming intermediates can overcome the molecular bottlenecks to acquire pluripotency. To decipher the underlying decisive factors driving cell fate, we identified induced pluripotent stem cells or stromal-like cells (iPSCs/SLCs) and iPSCs or trophoblast-like cells (iPSCs/TLCs) fate bifurcations by reconstructing cellular trajectory. The mesenchymal-epithelial transition and the activation of pluripotency networks are the main molecular series in successful reprogramming. Correspondingly, intermediates diverge into SLCs accompanied by the inhibition of cell cycle genes and the activation of extracellular matrix genes, whereas the TLCs fate is characterized by the up-regulation of placenta development genes. Combining putative gene regulatory networks, seven (Taf7, Ezh2, Klf2, etc.) and three key factors (Cdc5l, Klf4, and Nanog) were individually identified as drivers of the successful reprogramming by triggering downstream pluripotent networks during iPSCs/SLCs and iPSCs/TLCs fate bifurcation. Conversely, 11 factors (Cebpb, Sox4, Junb, etc.) and four factors (Gata2, Jund, Ctnnb1, etc.) drive SLCs fate and TLCs fate, respectively. Our study sheds new light on the understanding of decisive factors driving cell fate, which is helpful for improving reprogramming efficiency through manipulating cell fates to avoid alternative fates.

Root Cap to Soil Interface: A Driving Force Toward Plant Adaptation and Development.

Land plants have developed robust roots to grow in diverse soil ecosystems. The distal end of the root tip has a specialized organ called the 'root cap'. The root cap assists the roots in penetrating the ground, absorbing water and minerals, avoiding heavy metals and regulating the rhizosphere microbiota. Furthermore, root-cap-derived auxin governs the lateral root patterning and directs root growth under varying soil conditions. The root cap formation is hypothesized as one of the key innovations during root evolution. Morphologically diversified root caps in early land plant lineage and later in angiosperms aid in improving the adaptation of roots and, thereby, plants in diverse soil environments. This review article presents a retrospective view of the root cap's important morphological and physiological characteristics for the root-soil interaction and their response toward various abiotic and biotic stimuli. Recent single-cell RNAseq data shed light on root cap cell-type-enriched genes. We compiled root cap cell-type-enriched genes from Arabidopsis, rice, maize and tomato and analyzed their transcription factor (TF) binding site enrichment. Further, the putative gene regulatory networks derived from root-cap-enriched genes and their TF regulators highlight the species-specific biological functions of root cap genes across the four plant species.

Yield-associated putative gene regulatory networks in Oryza sativa L. subsp. indica and their association with high-yielding genotypes.

With the increase in population and economies of developing countries in Asia and Africa, the research towards securing future food demands is an imminent need. Among japonica and indica genotypes, indica rice varieties are largely cultivated across the globe. However, our present understanding of yield-contributing gene information stems mainly from japonica and studies on the yield potential of indica genotypes are limited. In the present study, yield contributing orthologous genes previously characterized from japonica varieties were identified in the indica genome and analysed with binGO tool for GO biological processes categorization. Transcription factor binding site enrichment analysis in the promoters of yield-related genes of indica was performed with MEME-AME tool that revealed putative common TF regulators are enriched in flower development, two-component signalling and water deprivation biological processes. Gene regulatory networks revealed important TF-target interactions that might govern yield-related traits. Some of the identified candidate genes were validated by qRT-PCR analysis for their expression and association with yield-related traits among 16 widely cultivated popular indica genotypes. Further, SNP-metabolite-trait association analysis was performed using high-yielding indica variety Rasi. This resulted in the identification of putative SNP variations in TF regulators and targeted yield genes significantly linked with metabolite accumulation. The study suggests some of the high yielding indica genotypes such as Ravi003, Rasi and Kavya could be used as potential donors in breeding programs based on yield gene expression analysis and SNP-metabolites associations.

Gene Regulatory Networks of Epidermal and Neural Fate Choice in a Chordate.

Neurons are a highly specialized cell type only found in metazoans. They can be scattered throughout the body or grouped together, forming ganglia or nerve cords. During embryogenesis, centralized nervous systems develop from the ectoderm, which also forms the epidermis. How pluripotent ectodermal cells are directed toward neural or epidermal fates, and to which extent this process is shared among different animal lineages, are still open questions. Here, by using micromere explants, we were able to define in silico the putative gene regulatory networks (GRNs) underlying the first steps of the epidermis and the central nervous system formation in the cephalochordate amphioxus. We propose that although the signal triggering neural induction in amphioxus (i.e., Nodal) is different from vertebrates, the main transcription factors implicated in this process are conserved. Moreover, our data reveal that transcription factors of the neural program seem to not only activate neural genes but also to potentially have direct inputs into the epidermal GRN, suggesting that the Nodal signal might also contribute to neural fate commitment by repressing the epidermal program. Our functional data on whole embryos support this result and highlight the complex interactions among the transcription factors activated by the signaling pathways that drive ectodermal cell fate choice in chordates.

Sonic hedgehog specifies flight feather positional information in avian wings.

ABSTRACTClassical tissue recombination experiments performed in the chick embryo provide evidence that signals operating during early limb development specify the position and identity of feathers. Here, we show that Sonic hedgehog (Shh) signalling in the embryonic chick wing bud specifies positional information required for the formation of adult flight feathers in a defined spatial and temporal sequence that reflects their different identities. We also reveal that Shh signalling is interpreted into specific patterns of Sim1 and Zic transcription factor expression, providing evidence of a putative gene regulatory network operating in flight feather patterning. Our data suggest that flight feather specification involved the co-option of the pre-existing digit patterning mechanism and therefore uncovers an embryonic process that played a fundamental step in the evolution of avian flight.

The acquisition of positional information across the radial axis of the cochlea.

The mammalian cochlea detects sound and transmits this information to the brain. A cross section through the cochlea reveals functionally distinct epithelial domains arrayed around the circumference of a fluid-filled duct. Six major domains include two on the roof of the duct (Reissner's membrane medially and the stria vascularis laterally) and four across the floor of the duct, including the medial and lateral halves of the sensory domain, the organ of Corti. These radial domains are distinguishable in the embryonic cochlea by differential expression of transcription factors, and we focus here on a subset of the factors that can influence cochlear fates. We then move upstream of these genes to identify which of five signaling pathways (Notch, Fgf, Wnt, Bmp, and Shh) controls their spatial patterns of expression. We link the signaling pathways to their downstream genes, separating them by their radial position, to create putative gene regulatory networks (GRNs) from two time points, before and during the time when six radial compartments arise. These GRNs offer a framework for understanding the acquisition of positional information across the radial axis of the cochlea, and to guide therapeutic approaches to repair or regenerate distinct cochlear components that may contribute to hearing loss.

The genetic architecture of type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1D) is a complex autoimmune disorder characterised by loss of the insulin-producing pancreatic beta cells in genetically predisposed individuals, ultimately resulting in insulin deficiency and hyperglycaemia. T1D is most common among children and young adults, and the incidence is on the rise across the world. The aetiology of T1D is hypothesized to involve genetic and environmental factors that result in the T-cell mediated destruction of pancreatic beta cells. There is a strong genetic risk to T1D; with genome-wide association studies (GWAS) identifying over 60 susceptibility regions within the human genome which are marked by single nucleotide polymorphisms (SNPs). Here, we review what is currently known about the genetics of T1D. We argue that advancing our understanding of the aetiology and pathogenesis of T1D will require the integration of genome biology (omics-data) with GWAS data, thereby making it possible to elucidate the putative gene regulatory networks modulated by disease-associated SNPs. This approach has a potential to revolutionize clinical management of T1D in an era of precision medicine.

Interplay between cardiac transcription factors and non-coding RNAs in predisposing to atrial fibrillation.

There is growing evidence that putative gene regulatory networks including cardio-enriched transcription factors, such as PITX2, TBX5, ZFHX3, and SHOX2, and their effector/target genes along with downstream non-coding RNAs can play a potentially important role in the process of adaptive and maladaptive atrial rhythm remodeling. In turn, expression of atrial fibrillation-associated transcription factors is under the control of upstream regulatory non-coding RNAs. This review broadly explores gene regulatory mechanisms associated with susceptibility to atrial fibrillation-with key examples from both animal models and patients-within the context of both cardiac transcription factors and non-coding RNAs. These two systems appear to have multiple levels of cross-regulation and act coordinately to achieve effective control of atrial rhythm effector gene expression. Perturbations of a dynamic expression balance between transcription factors and corresponding non-coding RNAs can provoke the development or promote the progression of atrial fibrillation. We also outline deficiencies in current models and discuss ongoing studies to clarify remaining mechanistic questions. An understanding of the function of transcription factors and non-coding RNAs in gene regulatory networks associated with atrial fibrillation risk will enable the development of innovative therapeutic strategies.

Gene expression profiling and construction of a putative gene regulatory network of bladder cancer tumor-initiating cells.

Human bladder cancer tumors have been shown to contain a subpopulation of cells with stem-like characteristics that may trigger tumor growth, recurrence, and metastasis. These cells, known as tumor-initiating cells (TICs), would be effective diagnostic tools and valuable therapeutic targets. Here, we report the isolation of TICs from seven bladder cancer cell lines and show that TICs from different sources vary on their ability to form tumorspheres in vitro and generate xenografts in vivo, which suggest they are remarkably heterogeneous. We used the Affymetrix PrimeView™ Human Gene Expression Array to analyze gene expression profiles of bladder TICs, which may help understand their tumorigenic capacities and develop novel treatments specifically targeted toward these cells. We then constructed a transcription factor-gene regulatory network that includes three key transcription factors that are involved in cell survival, differentiation, proliferation, and apoptosis. We validated our findings by analyzing mRNA expression of the key genes in this network in 24 clinical tissues. Our results suggest that this transcription factor-gene regulatory network could be useful in the development of clinical diagnostic tools and therapy approaches for bladder cancer.

Developing an ancient epithelial appendage: FGF signalling regulates early tail denticle formation in sharks

BackgroundVertebrate epithelial appendages constitute a diverse group of organs that includes integumentary structures such as reptilian scales, avian feathers and mammalian hair. Recent studies have provided new evidence for the homology of integumentary organ development throughout amniotes, despite their disparate final morphologies. These structures develop from conserved molecular signalling centres, known as epithelial placodes. It is not yet certain whether this homology extends beyond the integumentary organs of amniotes, as there is a lack of knowledge regarding their development in basal vertebrates. As the ancient sister lineage of bony vertebrates, extant chondrichthyans are well suited to testing the phylogenetic depth of this homology. Elasmobranchs (sharks, skates and rays) possess hard, mineralised epithelial appendages called odontodes, which include teeth and dermal denticles (placoid scales). Odontodes constitute some of the oldest known vertebrate integumentary appendages, predating the origin of gnathostomes. Here, we used an emerging model shark (Scyliorhinus canicula) to test the hypothesis that denticles are homologous to other placode-derived amniote integumentary organs. To examine the conservation of putative gene regulatory network (GRN) member function, we undertook small molecule inhibition of fibroblast growth factor (FGF) signalling during caudal denticle formation.ResultsWe show that during early caudal denticle morphogenesis, the shark expresses homologues of conserved developmental gene families, known to comprise a core GRN for early placode morphogenesis in amniotes. This includes conserved expression of FGFs, sonic hedgehog (shh) and bone morphogenetic protein 4 (bmp4). Additionally, we reveal that denticle placodes possess columnar epithelial cells with a reduced rate of proliferation, a conserved characteristic of amniote skin appendage development. Small molecule inhibition of FGF signalling revealed placode development is FGF dependent, and inhibiting FGF activity resulted in downregulation of shh and bmp4 expression, consistent with the expectation from comparison to the amniote integumentary appendage GRN.ConclusionOverall, these findings suggest the core GRN for building vertebrate integumentary epithelial appendages has been highly conserved over 450 million years. This provides evidence for the continuous, historical homology of epithelial appendage placodes throughout jawed vertebrates, from sharks to mammals. Epithelial placodes constitute the shared foundation upon which diverse vertebrate integumentary organs have evolved.

Analysis of global gene expression profiles during the flowering initiation process of Lilium × formolongi.

The onset of flowering is critical for the reproductive development of plants. Lilium × formolongi is a lily hybrid that flowers within a year after sowing. We successfully identified four important stages during vegetative growth and flowering initiation of L. × formolongi under long day conditions. The plant tissues from the four stages were used in a genome-wide transcriptional analysis to investigate stage-specific changes of gene expression in L. × formolongi. In total, the sequence reads of the four RNA-sequencing libraries were assembled into 52,824 unigenes, of which 37,031 (70.10%) were differentially expressed. The global expression dynamics of the differentially expressed genes were predominant in flowering induction phase I and the floral differentiation stage, but down-regulated in flowering induction phase II. Various transcription factor families relevant to flowering were elucidated, and the members of the MADS-box, SBP and CO-like transcription factor families were the most represented. There were 85 differentially expressed genes relevant to flowering. CONSTANS-LIKE, FLOWERING LOCUS T, TREHALOSE-6-PHOSPHATE SYNTHASE and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE homologs were discovered and may play significant roles in the flowering induction and transition process of L. × formolongi. A putative gene regulatory network, including photoperiod, age-dependent and trehalose-6-phosphate flowering pathways, was constructed. This is the first expression dataset obtained from a transcriptome analysis of photoperiod-mediated flowering pathway in lily, and it is valuable for the exploration of the molecular mechanisms of flowering initiation and the short vegetative stage of L. × formolongi.

Migrating into Genomics with the Neural Crest

Neural crest cells are a fascinating embryonic cell type, unique to vertebrates, which arise within the central nervous system but emigrate soon after its formation and migrate to numerous and sometimes distant locations in the periphery. Following their migratory phase, they differentiate into diverse derivatives ranging from peripheral neurons and glia to skin melanocytes and craniofacial cartilage and bone. The molecular underpinnings underlying initial induction of prospective neural crest cells at the neural plate border to their migration and differentiation have been modeled in the form of a putative gene regulatory network. This review describes experiments performed in my laboratory in the past few years aimed to test and elaborate this gene regulatory network from both an embryonic and evolutionary perspective. The rapid advances in genomic technology in the last decade have greatly expanded our knowledge of important transcriptional inputs and epigenetic influences on neural crest development. The results reveal new players and new connections in the neural crest gene regulatory network and suggest that it has an ancient origin at the base of the vertebrate tree.

Inference of sparse combinatorial-control networks from gene-expression data: a message passing approach

BackgroundTranscriptional gene regulation is one of the most important mechanisms in controlling many essential cellular processes, including cell development, cell-cycle control, and the cellular response to variations in environmental conditions. Genes are regulated by transcription factors and other genes/proteins via a complex interconnection network. Such regulatory links may be predicted using microarray expression data, but most regulation models suppose transcription factor independence, which leads to spurious links when many genes have highly correlated expression levels.ResultsWe propose a new algorithm to infer combinatorial control networks from gene-expression data. Based on a simple model of combinatorial gene regulation, it includes a message-passing approach which avoids explicit sampling over putative gene-regulatory networks. This algorithm is shown to recover the structure of a simple artificial cell-cycle network model for baker's yeast. It is then applied to a large-scale yeast gene expression dataset in order to identify combinatorial regulations, and to a data set of direct medical interest, namely the Pleiotropic Drug Resistance (PDR) network.ConclusionsThe algorithm we designed is able to recover biologically meaningful interactions, as shown by recent experimental results [1]. Moreover, new cases of combinatorial control are predicted, showing how simple models taking this phenomenon into account can lead to informative predictions and allow to extract more putative regulatory interactions from microarray databases.

STAR NET 2: a web-based tool for accelerating discovery of gene regulatory networks using microarray co-expression data

BackgroundAlthough expression microarrays have become a standard tool used by biologists, analysis of data produced by microarray experiments may still present challenges. Comparison of data from different platforms, organisms, and labs may involve complicated data processing, and inferring relationships between genes remains difficult.ResultsSTARNET 2 is a new web-based tool that allows post hoc visual analysis of correlations that are derived from expression microarray data. STARNET 2 facilitates user discovery of putative gene regulatory networks in a variety of species (human, rat, mouse, chicken, zebrafish, Drosophila, C. elegans, S. cerevisiae, Arabidopsis and rice) by graphing networks of genes that are closely co-expressed across a large heterogeneous set of preselected microarray experiments. For each of the represented organisms, raw microarray data were retrieved from NCBI's Gene Expression Omnibus for a selected Affymetrix platform. All pairwise Pearson correlation coefficients were computed for expression profiles measured on each platform, respectively. These precompiled results were stored in a MySQL database, and supplemented by additional data retrieved from NCBI. A web-based tool allows user-specified queries of the database, centered at a gene of interest. The result of a query includes graphs of correlation networks, graphs of known interactions involving genes and gene products that are present in the correlation networks, and initial statistical analyses. Two analyses may be performed in parallel to compare networks, which is facilitated by the new HEATSEEKER module.ConclusionSTARNET 2 is a useful tool for developing new hypotheses about regulatory relationships between genes and gene products, and has coverage for 10 species. Interpretation of the correlation networks is supported with a database of previously documented interactions, a test for enrichment of Gene Ontology terms, and heat maps of correlation distances that may be used to compare two networks. The list of genes in a STARNET network may be useful in developing a list of candidate genes to use for the inference of causal networks. The tool is freely available at , and does not require user registration.