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Background: Cis-regulatory elements (CREs) are regions of DNA that regulate the expression of nearby genes. Changes in these elements can lead to phenotypic variations and adaptations in different populations. However, the regulatory dynamics underlying the local adaptation of traits remain poorly understood in Chinese and Western pigs. By comparing the chromatin accessibility profiles of skeletal muscle, liver, and fat between these two pig populations, we aimed to identify key regulatory elements that could explain phenotypic differences observed between the two groups. Results: Our results revealed that the genome-wide chromatin accessibility profiles were largely similar at a qualitative level within tissues. However, we also identified local regions that exhibited quantitative differences, most of which occurred in liver tissue. Interestingly, we found that most of the increased chromatin accessibility in the livers of Chinese pigs was associated with tissue-specific openness. Furthermore, we observed a positive correlation between the ATAC-seq signal at the transcript start site (TSS) and the expression levels of nearby genes. Motif enrichment analysis revealed NR2F1 as a key regulator in Chinese pigs. Differentially expressed genes (DEGs) in Chinese pigs showed enrichment for NR2F1 response targets. One of the genes regulated by NR2F1 in Chinese pigs, NPC1 , harbored a high alternative allelic frequency in the intron region. Conclusion: Overall, our study provides valuable insights into the regulatory dynamics underlying phenotypic variation in pigs. By elucidating the role of CREs in driving phenotypic variation, we can better understand the genetic basis of complex traits and potentially identify targets for genetic improvement in livestock breeding programs. [ABSTRACT FROM AUTHOR]

Background: The relationship between epigenomics and plant specialised metabolism remains largely unexplored despite the fundamental importance of epigenomics in gene regulation and, potentially, yield of products of plant specialised metabolic pathways. The glandular trichomes of Cannabis sativa are an emerging model system that produce large quantities of cannabinoid and terpenoid specialised metabolites with known medicinal and commercial value. To address this lack of epigenomic data, we mapped H3K4 trimethylation, H3K56 acetylation, H3K27 trimethylation post-translational modifications and the histone variant H2A.Z, using chromatin immunoprecipitation, in C. sativa glandular trichomes, leaf, and stem tissues. Corresponding transcriptomic (RNA-seq) datasets were integrated, and tissue-specific analyses conducted to relate chromatin states to glandular trichome specific gene expression. Results: The promoters of cannabinoid and terpenoid biosynthetic genes, specialised metabolite transporter genes, defence related genes, and starch and sucrose metabolism were enriched specifically in trichomes for histone marks H3K4me3 and H3K56ac, consistent with active transcription. We identified putative trichome-specific enhancer elements by identifying intergenic regions of H3K56ac enrichment, a histone mark that maintains enhancer accessibility, then associated these to putative target genes using the tissue specific gene transcriptomic data. Bi-valent chromatin loci specific to glandular trichomes, marked with H3K4 trimethylation and H3K27 trimethylation, were associated with genes of MAPK signalling pathways and plant specialised metabolism pathways, supporting recent hypotheses that implicate bi-valent chromatin in plant defence. The histone variant H2A.Z was largely found in intergenic regions and enriched in chromatin that contained genes involved in DNA homeostasis. Conclusion: We report the first genome-wide histone post-translational modification maps for C. sativa glandular trichomes, and more broadly for glandular trichomes in plants. Our findings have implications in plant adaptation and stress responses and provide a basis for enhancer-mediated, targeted, gene transformation studies in plant glandular trichomes. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) and inflammatory stress responses contribute to islet dysfunction in type 2 diabetes (T2D). Comprehensive genomic understanding of these human islet stress responses and whether T2D-associated genetic variants modulate them is lacking. Here, comparative transcriptome and epigenome analyses of human islets exposed ex vivo to these stressors revealed 30% of expressed genes and 14% of islet cis -regulatory elements (CREs) as stress responsive, modulated largely in an ER- or cytokine-specific fashion. T2D variants overlapped 86 stress-responsive CREs, including 21 induced by ER stress. We linked the rs6917676-T T2D risk allele to increased islet ER-stress-responsive CRE accessibility and allele-specific β cell nuclear factor binding. MAP3K5 , the ER-stress-responsive putative rs6917676 T2D effector gene, promoted stress-induced β cell apoptosis. Supporting its pro-diabetogenic role, MAP3K5 expression correlated inversely with human islet β cell abundance and was elevated in T2D β cells. This study provides genome-wide insights into human islet stress responses and context-specific T2D variant effects. [Display omitted] • Genome-wide comparative human islet ER/cytokine stress response maps • T2D variants overlap 86 stress-responsive cis -regulatory elements (CREs) • Variant-to-function links between T2D variant rs6917676, MAP3K5 , and islet apoptosis • Risk allele increased ER-stress-induced CRE accessibility and MAP3K5 -mediated death This multi-omic study dissects the genome-wide effects of ER stress and pro-inflammatory cytokines on human islet transcriptional regulatory networks and motivates variant-to-function studies linking the rs6917676 T2D risk allele to enhanced ER-stress-responsive MAP3K5- mediated β cell apoptosis. The findings suggest that MAP3K5 inhibitors (e.g., selonsertib) could therapeutically reduce stress-induced β cell apoptosis. [ABSTRACT FROM AUTHOR]

Viruses sense cellular states and environmental signals by recruiting host transcription factors to cis -regulatory elements (CREs) in viral genomes. Viral CREs act as signal integration hubs that impart specificity to viral gene regulation. Viral CREs are highly evolvable due to their high mutation rate and ability to rapidly change sensing mechanisms, affecting infection spread and patient outcomes. Latent viral infections can be treated by targeting viral CREs for reactivation or permanent silencing. To withstand a hostile cellular environment and replicate, viruses must sense, interpret, and respond to many internal and external cues. Retroviruses and DNA viruses can intercept these cues impinging on host transcription factors via cis -regulatory elements (CREs) in viral genomes, allowing them to sense and coordinate context-specific responses to varied signals. Here, we explore the characteristics of viral CREs, the classes of signals and host transcription factors that regulate them, and how this informs outcomes of viral replication, immune evasion, and latency. We propose that viral CREs constitute central hubs for signal integration from multiple pathways and that sequence variation between viral isolates can rapidly rewire sensing mechanisms, contributing to the variability observed in patient outcomes. [ABSTRACT FROM AUTHOR]

Abstract Many species of Sapindaceae, such as lychee, longan, and rambutan, provide nutritious and delicious fruit. Understanding the molecular genetic mechanisms that underlie the regulation of flowering is essential for securing flower and fruit productivity. Most endogenous and exogenous flowering cues are integrated into the florigen encoded by FLOWERING LOCUS T. However, the regulatory mechanisms of flowering remain poorly understood in Sapindaceae. Here, we identified 60 phosphatidylethanolamine-binding protein-coding genes from six Sapindaceae plants. Gene duplication events led to the emergence of two or more paralogs of the FT gene that have evolved antagonistic functions in Sapindaceae. Among them, the FT1-like genes are functionally conserved and promote flowering, while the FT2-like genes likely serve as repressors that delay flowering. Importantly, we show here that the natural variation at nucleotide position − 1437 of the lychee FT1 promoter determined the binding affinity of the SVP protein (LcSVP9), which was a negative regulator of flowering, resulting in the differential expression of LcFT1, which in turn affected flowering time in lychee. This finding provides a potential molecular marker for breeding lychee. Taken together, our results reveal some crucial aspects of FT gene family genetics that underlie the regulation of flowering in Sapindaceae.

Abstract Background The endothelial-to-hematopoietic transition (EHT) process during definitive hematopoiesis is highly conserved in vertebrates. Stage-specific expression of transposable elements (TEs) has been detected during zebrafish EHT and may promote hematopoietic stem cell (HSC) formation by activating inflammatory signaling. However, little is known about how TEs contribute to the EHT process in human and mouse. Results We reconstructed the single-cell EHT trajectories of human and mouse and resolved the dynamic expression patterns of TEs during EHT. Most TEs presented a transient co-upregulation pattern along the conserved EHT trajectories, coinciding with the temporal relaxation of epigenetic silencing systems. TE products can be sensed by multiple pattern recognition receptors, triggering inflammatory signaling to facilitate HSC emergence. Interestingly, we observed that hypoxia-related signals were enriched in cells with higher TE expression. Furthermore, we constructed the hematopoietic cis-regulatory network of accessible TEs and identified potential TE-derived enhancers that may boost the expression of specific EHT marker genes. Conclusions Our study provides a systematic vision of how TEs are dynamically controlled to promote the hematopoietic fate decisions through transcriptional and cis-regulatory networks, and pre-train the immunity of nascent HSCs.

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Many species of Sapindaceae, such as lychee, longan, and rambutan, provide nutritious and delicious fruit. Understanding the molecular genetic mechanisms that underlie the regulation of flowering is essential for securing flower and fruit productivity. Most endogenous and exogenous flowering cues are integrated into the florigen encoded by FLOWERING LOCUS T. However, the regulatory mechanisms of flowering remain poorly understood in Sapindaceae. Here, we identified 60 phosphatidylethanolamine-binding protein-coding genes from six Sapindaceae plants. Gene duplication events led to the emergence of two or more paralogs of the FT gene that have evolved antagonistic functions in Sapindaceae. Among them, the FT1-like genes are functionally conserved and promote flowering, while the FT2-like genes likely serve as repressors that delay flowering. Importantly, we show here that the natural variation at nucleotide position − 1437 of the lychee FT1 promoter determined the binding affinity of the SVP protein (LcSVP9), which was a negative regulator of flowering, resulting in the differential expression of LcFT1, which in turn affected flowering time in lychee. This finding provides a potential molecular marker for breeding lychee. Taken together, our results reveal some crucial aspects of FT gene family genetics that underlie the regulation of flowering in Sapindaceae. [ABSTRACT FROM AUTHOR]

Epigenetic changes serve as a cellular memory for cumulative cold recognition in both herbaceous and tree species, including bud dormancy. However, most studies have discussed predicted chromatin structure with respect to histone marks. In the present study, we investigated the structural dynamics of bona fide chromatin to determine how plants recognize prolonged chilling during the initial stage of bud dormancy. The vegetative axillary buds of the 'Fuji' apple, which shows typical low temperature-dependent, but not photoperiod, dormancy induction, were used for the chromatin structure and transcriptional change analyses. The results were integrated using a deep-learning model and interpreted using statistical models, including Bayesian estimation. Although our model was constructed using a small dataset of two time points, chromatin remodelling due to random changes was excluded. The involvement of most nucleosome structural changes in transcriptional changes and the pivotal contribution of cold-driven circadian rhythm-dependent pathways regulated by the mobility of cis -regulatory elements were predicted. These findings may help to develop potential genetic targets for breeding species with less bud dormancy to overcome the effects of short winters during global warming. Our artificial intelligence concept can improve epigenetic analysis using a small dataset, especially in non-model plants with immature genome databases. [ABSTRACT FROM AUTHOR]

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation. [ABSTRACT FROM AUTHOR]

Background: The endothelial-to-hematopoietic transition (EHT) process during definitive hematopoiesis is highly conserved in vertebrates. Stage-specific expression of transposable elements (TEs) has been detected during zebrafish EHT and may promote hematopoietic stem cell (HSC) formation by activating inflammatory signaling. However, little is known about how TEs contribute to the EHT process in human and mouse. Results: We reconstructed the single-cell EHT trajectories of human and mouse and resolved the dynamic expression patterns of TEs during EHT. Most TEs presented a transient co-upregulation pattern along the conserved EHT trajectories, coinciding with the temporal relaxation of epigenetic silencing systems. TE products can be sensed by multiple pattern recognition receptors, triggering inflammatory signaling to facilitate HSC emergence. Interestingly, we observed that hypoxia-related signals were enriched in cells with higher TE expression. Furthermore, we constructed the hematopoietic cis-regulatory network of accessible TEs and identified potential TE-derived enhancers that may boost the expression of specific EHT marker genes. Conclusions: Our study provides a systematic vision of how TEs are dynamically controlled to promote the hematopoietic fate decisions through transcriptional and cis-regulatory networks, and pre-train the immunity of nascent HSCs. [ABSTRACT FROM AUTHOR]

BackgroundCis-regulatory elements (CREs) are regions of DNA that regulate the expression of nearby genes. Changes in these elements can lead to phenotypic variations and adaptations in different populations. However, the regulatory dynamics underlying the local adaptation of traits remain poorly understood in Chinese and Western pigs. By comparing the chromatin accessibility profiles of skeletal muscle, liver, and fat between these two pig populations, we aimed to identify key regulatory elements that could explain phenotypic differences observed between the two groups.ResultsOur results revealed that the genome-wide chromatin accessibility profiles were largely similar at a qualitative level within tissues. However, we also identified local regions that exhibited quantitative differences, most of which occurred in liver tissue. Interestingly, we found that most of the increased chromatin accessibility in the livers of Chinese pigs was associated with tissue-specific openness. Furthermore, we observed a positive correlation between the ATAC-seq signal at the transcript start site (TSS) and the expression levels of nearby genes. Motif enrichment analysis revealed NR2F1 as a key regulator in Chinese pigs. Differentially expressed genes (DEGs) in Chinese pigs showed enrichment for NR2F1 response targets. One of the genes regulated by NR2F1 in Chinese pigs, NPC1, harbored a high alternative allelic frequency in the intron region.ConclusionOverall, our study provides valuable insights into the regulatory dynamics underlying phenotypic variation in pigs. By elucidating the role of CREs in driving phenotypic variation, we can better understand the genetic basis of complex traits and potentially identify targets for genetic improvement in livestock breeding programs.

Recent evidence suggests that human gene promoters display gene expression regulatory mechanisms beyond the typical single gene local transcription modulation. In mammalian genomes, genes with an associated bidirectional promoter are abundant; bidirectional promoter architecture serves as a regulatory hub for a gene pair expression. However, it has been suggested that its contribution to transcriptional regulation might exceed local transcription initiation modulation. Despite their abundance, the functional consequences of bidirectional promoter architecture remain largely unexplored. This work studies the long-range gene expression regulatory role of a long non-coding RNA gene promoter using chromosome conformation capture methods. We found that this particular bidirectional promoter contributes to distal gene expression regulation in a target-specific manner by establishing promoter–promoter interactions. In particular, we validated that the promoter–promoter interactions of this regulatory element with the promoter of distal gene BBX contribute to modulating the transcription rate of this gene; removing the bidirectional promoter from its genomic context leads to a rearrangement of BBX promoter–enhancer interactions and to increased gene expression. Moreover, long-range regulatory functionality is not directly dependent on its associated non-coding gene pair expression levels. [ABSTRACT FROM AUTHOR]

Background: Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Methods: Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An a-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the a-globin domain in erythroid chicken cells. Results: Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An a-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over aD and aA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that a-globin CRE-2 is required for the induction of aD and aA. Analysis of TF binding motifs at a-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Conclusion: Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells. [ABSTRACT FROM AUTHOR]

AbstractThe "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

BackgroundCis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis.MethodsAnalysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells.ResultsErythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding.ConclusionOur findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

The medicinal plant Spatholobus suberectus Dunn is easily exposed to shade stress during growth, but its shade responses and shade stress resistant mechanisms have not been clarified. In this study, shade treatments including four attenuated sunlight intensities (100%, 60%, 40%, and 10%) and three shade durations (30 d, 45 d, and 60 d) were applied to S. suberectus. The shade-induced morphological indicators, phytohormonal regulations, metabolic flavonoids contents, transcriptomic flavonoid pathway gene expressions, and stress physiological changes of S. suberectus were analyzed. The putative promoter cis-regulatory elements (CREs) of 18 flavonoid biosynthetic pathway genes were identified. Results showed the stem growth indicators of S. suberectus were better at 40% light intensity. Phytohormones were involved in the shade-induced responses. Short-term shade (30 d) increased total flavonoids, gallated catechins and especially epigallocatechin gallate contents and favored for boosting medicinal value. Long-term shade (45 d, 60 d) tended to decrease flavonoids. The shade-induced flavonoids changes were attributed to their corresponding biosynthesizing genes expression variations. The high antioxidant capacity and the presence of phytohormone-, stress-, and development-related CREs provided the basis for stress resistance. In conclusion, the multiple responses under shade and the CREs analysis elucidated S. suberectus’ shade tolerance.

Throughout embryonic development, the shaping of the functional and morphological characteristics of embryos is orchestrated by an intricate interaction between transcription factors and cis-regulatory elements. In this study, we conducted a comprehensive analysis of deuterostome cis-regulatory landscapes during gastrulation, focusing on four paradigmatic species: the echinoderm Strongylocentrotus purpuratus, the cephalochordate Branchiostoma lanceolatum, the urochordate Ciona intestinalis, and the vertebrate Danio rerio. Our approach involved comparative computational analysis of ATAC-seq datasets to explore the genome-wide blueprint of conserved transcription factor binding motifs underlying gastrulation. We identified a core set of conserved DNA binding motifs associated with 62 known transcription factors, indicating the remarkable conservation of the gastrulation regulatory landscape across deuterostomes. Our findings offer valuable insights into the evolutionary molecular dynamics of embryonic development, shedding light on conserved regulatory subprograms and providing a comprehensive perspective on the conservation and divergence of gene regulation underlying the gastrulation process.

MicroRNAs (miRNAs) play important roles in plant responses to abiotic stress. Numerous studies have been increasing in respect to miRNA identification under stress conditions. In this study we analysed the expression patterns of seven miRNAs (miR156, 159, 169, 396, 397, 398 and 399) from cadmium stressed seedlings of Finger millet by RT-qPCR. Further these miRNAs were cloned and sequenced, which conformed its presence. Predicted targets and GO analysis of the miRNAs were found to be involved in diverse cellular processes in plants, development, apoptosis, detoxification, catalysis, protein modification. Cis-regulatory elements identification suggested their involvement in regulatory networks. This is the first study to demonstrate differentially expressed miRNA in Finger millet under cadmium stress. Findings in the present study prominence the role played by miRNAs in Finger millet under cadmium stress.

Abstract Background Prioritizing genes that underlie complex brain disorders poses a considerable challenge. Despite previous studies have found that they shared symptoms and heterogeneity, it remained difficult to systematically identify the risk genes associated with them. Methods By using the CAGE (Cap Analysis of Gene Expression) read alignment files for 439 human cell and tissue types (including primary cells, tissues and cell lines) from FANTOM5 project, we predicted enhancer-promoter interactions (EPIs) of 439 cell and tissue types in human, and examined their reliability. Then we evaluated the genetic heritability of 17 diverse brain disorders and behavioral-cognitive phenotypes in each neural cell type, brain region, and developmental stage. Furthermore, we prioritized genes associated with brain disorders and phenotypes by leveraging the EPIs in each neural cell and tissue type, and analyzed their pleiotropy and functionality for different categories of disorders and phenotypes. Finally, we characterized the spatiotemporal expression dynamics of these associated genes in cells and tissues. Results We found that identified EPIs showed activity specificity and network aggregation in cell and tissue types, and enriched TF binding in neural cells played key roles in synaptic plasticity and nerve cell development, i.e., EGR1 and SOX family. We also discovered that most neurological disorders exhibit heritability enrichment in neural stem cells and astrocytes, while psychiatric disorders and behavioral-cognitive phenotypes exhibit enrichment in neurons. Furthermore, our identified genes recapitulated well-known risk genes, which exhibited widespread pleiotropy between psychiatric disorders and behavioral-cognitive phenotypes (i.e., FOXP2), and indicated expression specificity in neural cell types, brain regions, and developmental stages associated with disorders and phenotypes. Importantly, we showed the potential associations of brain disorders with brain regions and developmental stages that have not been well studied. Conclusions Overall, our study characterized the gene-enhancer regulatory networks and genetic mechanisms in the human neural cells and tissues, and illustrated the value of reanalysis of publicly available genomic datasets.

The GARP transcription factors have been identified for multiple biological functions throughout the life cycle of a plant. Despite of its involvement in crucial functions, systematic study of GARPs remains obscure in plants. In this study, we explored the genomic, molecular, and evolutionary perspectives of the GARP gene family in the three major leguminous plants, namely chickpea (Cicer arietinum), soybean (Glycine max), and barrel clover (Medicago truncatula). Here, we identified 53, 56, and 107 GARP genes in Cicer, Medicago, and Glycine, respectively. They were classified into four clades and two sub-clades as per phylogenetic analysis, and the result was supported by consensus motifs, domain organization, and exon–intron structures. Detailed comparative analysis indicates conservation of the GARP gene family in plants. Identification of paralogous and orthologous gene pairs revealed that the expansion of this family occurs mainly through genome duplication in legumes. Additionally, the three-dimensional structure and functional enrichment analysis indicated their major role in signaling, growth, development, and stress processes. The chickpea GARP genes were also characterized for their transcript modulation in diverse plant organs and during pathogenic stress. Differential regulation of 24 CaGARP genes was observed during Ascochyta Blight (AB) stress. Characterization of AB-responsive genes reveals an over-representation of stress and hormone-binding elements on the promoter of CaGARPs. Additionally, interactome analysis also confirms the role of GARPs in plant stress and development. Our findings not only provide a handful of stress-responsive genes but also lay the foundation for prospective functional studies of GARPs in legumes. [ABSTRACT FROM AUTHOR]

MicroRNA(miRNA), a small non-coding class of RNA that regulates the gene expression, is conserved among several plant species. In the present study, an in-silico approach was adopted to identify miRNA from the known expressed sequences tags (ESTs) of Citrus reticulata L. A total of 17 miRNAs from 23 different ESTs along with their secondary structures and targets were predicted. The identified 63 targets include several transcription factors, proteins that regulate plant growth, development, flowering, and seed development together with stress response. The cis-regulatory element present at the promoter region of the MIR genes of C. reticulata describes their role during light responsiveness, auxin, gibberellins, abscisic acid (ABA), anthocyanin responsiveness, salicylic acid responsiveness, anaerobic induction, circadian control, nitrate dependent regulation of the cell cycle and DNA replication, defense, and stress responsiveness. The present study identifies the miRNAs along with their regulatory elements in C. reticulate. The study will also be useful for the research on identification from the genomic data of different plants and prediction of expression probability of identified MIR genes based on the presence of upstream promoter and other regulatory elements. [ABSTRACT FROM AUTHOR]

Abstract Background The auxin indole-3-acetic acid (IAA) is a vital phytohormone that influences plant growth and development. Our previous work showed that IAA content decreased during flower development in the medicinally important orchid Dendrobium officinale, while Aux/IAA genes were downregulated. However, little information about auxin-responsive genes and their roles in D. officinale flower development exists. Results This study validated 14 DoIAA and 26 DoARF early auxin-responsive genes in the D. officinale genome. A phylogenetic analysis classified the DoIAA genes into two subgroups. An analysis of cis-regulatory elements indicated that they were related by phytohormones and abiotic stresses. Gene expression profiles were tissue-specific. Most DoIAA genes (except for DoIAA7) were sensitive to IAA (10 μmol/L) and were downregulated during flower development. Four DoIAA proteins (DoIAA1, DoIAA6, DoIAA10 and DoIAA13) were mainly localized in the nucleus. A yeast two-hybrid assay showed that these four DoIAA proteins interacted with three DoARF proteins (DoARF2, DoARF17, DoARF23). Conclusions The structure and molecular functions of early auxin-responsive genes in D. officinale were investigated. The DoIAA-DoARF interaction may play an important role in flower development via the auxin signaling pathway.

Summary: The CRISPR‐Cas‐based genome editing field in plants is expanding rapidly. Editing plant promoters to obtain cis‐regulatory alleles with altered expression levels or patterns of target genes is a highly promising topic. However, primarily used CRISPR‐Cas9 has significant limitations when editing noncoding sequences like promoters, which have unique structures and regulatory mechanisms, including A‐T richness, repetitive redundancy, difficulty in identifying key regulatory regions, and a higher frequency of DNA structure, epigenetic modification, and protein binding accessibility issues. Researchers urgently require efficient and feasible editing tools and strategies to address these obstacles, enhance promoter editing efficiency, increase diversity in promoter polymorphism, and, most importantly, enable 'non‐silent' editing events that achieve precise target gene expression regulation. This article provides insights into the key challenges and references for implementing promoter editing‐based research in plants. [ABSTRACT FROM AUTHOR]

Background: Prioritizing genes that underlie complex brain disorders poses a considerable challenge. Despite previous studies have found that they shared symptoms and heterogeneity, it remained difficult to systematically identify the risk genes associated with them. Methods: By using the CAGE (Cap Analysis of Gene Expression) read alignment files for 439 human cell and tissue types (including primary cells, tissues and cell lines) from FANTOM5 project, we predicted enhancer-promoter interactions (EPIs) of 439 cell and tissue types in human, and examined their reliability. Then we evaluated the genetic heritability of 17 diverse brain disorders and behavioral-cognitive phenotypes in each neural cell type, brain region, and developmental stage. Furthermore, we prioritized genes associated with brain disorders and phenotypes by leveraging the EPIs in each neural cell and tissue type, and analyzed their pleiotropy and functionality for different categories of disorders and phenotypes. Finally, we characterized the spatiotemporal expression dynamics of these associated genes in cells and tissues. Results: We found that identified EPIs showed activity specificity and network aggregation in cell and tissue types, and enriched TF binding in neural cells played key roles in synaptic plasticity and nerve cell development, i.e., EGR1 and SOX family. We also discovered that most neurological disorders exhibit heritability enrichment in neural stem cells and astrocytes, while psychiatric disorders and behavioral-cognitive phenotypes exhibit enrichment in neurons. Furthermore, our identified genes recapitulated well-known risk genes, which exhibited widespread pleiotropy between psychiatric disorders and behavioral-cognitive phenotypes (i.e., FOXP2), and indicated expression specificity in neural cell types, brain regions, and developmental stages associated with disorders and phenotypes. Importantly, we showed the potential associations of brain disorders with brain regions and developmental stages that have not been well studied. Conclusions: Overall, our study characterized the gene-enhancer regulatory networks and genetic mechanisms in the human neural cells and tissues, and illustrated the value of reanalysis of publicly available genomic datasets. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play important roles in plant responses to abiotic stress. Numerous studies have been increasing in respect to miRNA identification under stress conditions. In this study we analysed the expression patterns of seven miRNAs (miR156, 159, 169, 396, 397, 398 and 399) from cadmium stressed seedlings of Finger millet by RT-qPCR. Further these miRNAs were cloned and sequenced, which conformed its presence. Predicted targets and GO analysis of the miRNAs were found to be involved in diverse cellular processes in plants, development, apoptosis, detoxification, catalysis, protein modification. Cis-regulatory elements identification suggested their involvement in regulatory networks. This is the first study to demonstrate differentially expressed miRNA in Finger millet under cadmium stress. Findings in the present study prominence the role played by miRNAs in Finger millet under cadmium stress. [ABSTRACT FROM AUTHOR]

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50–75% ammonium–sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment. [ABSTRACT FROM AUTHOR]

Co-option of transposable elements (TEs) to become part of existing or new enhancers is an important mechanism for evolution of gene regulation. However, contributions of lineage-specific TE insertions to recent regulatory adaptations remain poorly understood. Gibbons present a suitable model to study these contributions as they have evolved a lineage-specific TE called LAVA (LINE-AluSz-VNTR-Alu LIKE), which is still active in the gibbon genome. The LAVA retrotransposon is thought to have played a role in the emergence of the highly rearranged structure of the gibbon genome by disrupting transcription of cell cycle genes. In this study, we investigated whether LAVA may have also contributed to the evolution of gene regulation by adopting enhancer function. We characterized fixed and polymorphic LAVA insertions across multiple gibbons and found 96 LAVA elements overlapping enhancer chromatin states. Moreover, LAVA was enriched in multiple transcription factor binding motifs, was bound by an important transcription factor (PU.1), and was associated with higher levels of gene expression in cis We found gibbon-specific signatures of purifying/positive selection at 27 LAVA insertions. Two of these insertions were fixed in the gibbon lineage and overlapped with enhancer chromatin states, representing putative co-opted LAVA enhancers. These putative enhancers were located within genes encoding SETD2 and RAD9A, two proteins that facilitate accurate repair of DNA double-strand breaks and prevent chromosomal rearrangement mutations. Co-option of LAVA in these genes may have influenced regulation of processes that preserve genome integrity. Our findings highlight the importance of considering lineage-specific TEs in studying evolution of gene regulatory elements.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

Background: Brown planthopper (BPH; Nilaparvata lugens) is one of the most serious pests of rice in the world. Insect‐resistant genetic engineering is a very effective technology to control BPH. The promoters and cis‐regulatory elements inducible by plant‐feeding insects are critical for genetic engineering of insect‐resistant crops. Results: In this study, we cloned a promoter Ptps31 and a 7 bp cis–regulatory sequence that up‐regulated downstream genes induced by BPH feeding. The promoter of OsTPS31 (Ptps31) unresponsive to physical damage but responsive to BPH feeding was cloned and functionally verified. The results showed that expression of the OsBPH14 gene driven by the promoter region from −510 to −246 bp in rice could significantly improve the resistance to BPH. The promoter region from −376 to −370 bp (TAGTGTC) was identified as a cis‐regulatory sequence related to BPH feeding induction of downstream gene expression. Conclusion: The findings provide a new promoter and a new cis‐regulatory sequence tool for the research on and application of rice BPH resistance genes, as well as a new perspective for functional analysis of the OsTPS31 gene. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: The auxin indole-3-acetic acid (IAA) is a vital phytohormone that influences plant growth and development. Our previous work showed that IAA content decreased during flower development in the medicinally important orchid Dendrobium officinale, while Aux/IAA genes were downregulated. However, little information about auxin-responsive genes and their roles in D. officinale flower development exists. Results: This study validated 14 DoIAA and 26 DoARF early auxin-responsive genes in the D. officinale genome. A phylogenetic analysis classified the DoIAA genes into two subgroups. An analysis of cis-regulatory elements indicated that they were related by phytohormones and abiotic stresses. Gene expression profiles were tissue-specific. Most DoIAA genes (except for DoIAA7) were sensitive to IAA (10 μmol/L) and were downregulated during flower development. Four DoIAA proteins (DoIAA1, DoIAA6, DoIAA10 and DoIAA13) were mainly localized in the nucleus. A yeast two-hybrid assay showed that these four DoIAA proteins interacted with three DoARF proteins (DoARF2, DoARF17, DoARF23). Conclusions: The structure and molecular functions of early auxin-responsive genes in D. officinale were investigated. The DoIAA-DoARF interaction may play an important role in flower development via the auxin signaling pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: The fibroin heavy chain (FibH) is the core protein of silkworm silk, and a systematic analysis of its sequence (primary structure) is essential for understanding silkworm silk fibers. However, only a few FibH sequences have been identified. The recently published high-resolution silkworm pan-genome was useful in analyzing the FibH gene systematically. In this study, we extracted the complete FibH sequences of wild silkworms, local and improved strains from the silkworm pan-genome, bringing the total number of known silkworm FibH genes to 286. We characterized in detail the FibH variation during domestication and breeding and established a silkworm pedigree based on the FibH gene sequence. Additionally, we investigated the relationship between the cis-regulatory elements of FibH gene and silk yield in wild and domesticated silkworms. Our study laid the foundation for artificial silk as well as provided guidance for silkworm breeding. The highly repetitive and variable fibroin heavy chain (FibH) gene can be used as a silkworm identification; however, only a few complete FibH sequences are known. In this study, we extracted and examined 264 FibH gene complete sequences (FibHome) from a high-resolution silkworm pan-genome. The average FibH lengths of the wild silkworm, local, and improved strains were 19,698 bp, 16,427 bp, and 15,795 bp, respectively. All FibH sequences had a conserved 5′ and 3′ terminal non-repetitive (5′ and 3′ TNR, 99.74% and 99.99% identity, respectively) sequence and a variable repetitive core (RC). The RCs differed greatly, but they all shared the same motif. During domestication or breeding, the FibH gene mutated with hexanucleotide (GGTGCT) as the core unit. Numerous variations existed that were not unique to wild and domesticated silkworms. However, the transcriptional factor binding sites, such as fibroin modulator-binding protein, were highly conserved and had 100% identity in the FibH gene's intron and upstream sequences. The local and improved strains with the same FibH gene were divided into four families using this gene as a marker. Family I contained a maximum of 62 strains with the optional FibH (Opti-FibH, 15,960 bp) gene. This study provides new insights into FibH variations and silkworm breeding. [ABSTRACT FROM AUTHOR]

Organismal phenotypes result largely from inherited developmental programs, usually executed during embryonic and juvenile life stages. These programs are not blank slates onto which natural selection can draw arbitrary forms. Rather, the mechanisms of development play an integral role in shaping phenotypic diversity and help determine the evolutionary trajectories of species. Modern evolutionary biology must, therefore, account for these mechanisms in both theory and in practice. The gene regulatory network (GRN) concept represents a potent tool for achieving this goal whose utility has grown in tandem with advances in "omic" technologies and experimental techniques. However, while the GRN concept is widely utilized, it is often less clear what practical implications it has for conducting research in evolutionary developmental biology. In this Perspective, we attempt to provide clarity by discussing how experiments and projects can be designed in light of the GRN concept. We first map familiar biological notions onto the more abstract components of GRN models. We then review how diverse functional genomic approaches can be directed toward the goal of constructing such models and discuss current methods for functionally testing evolutionary hypotheses that arise from them. Finally, we show how the major steps of GRN model construction and experimental validation suggest generalizable workflows that can serve as a scaffold for project design. Taken together, the practical implications that we draw from the GRN concept provide a set of guideposts for studies aiming at unraveling the molecular basis of phenotypic diversity. [ABSTRACT FROM AUTHOR]

Changes in gene expression are a prominent feature of morphological evolution. These changes occur to hierarchical gene regulatory networks (GRNs) of transcription factor genes that regulate the expression of trait‐building differentiation genes. While changes in the expression of differentiation genes are essential to phenotypic evolution, they can be caused by mutations within cis‐regulatory elements (CREs) that drive their expression (cis‐evolution) or within genes for CRE‐interacting transcription factors (trans‐evolution). Locating these mutations remains a challenge, especially when experiments are limited to one species that possesses the ancestral or derived phenotype. We investigated CREs that control the expression of the differentiation genes tan and yellow, the expression of which evolved during the gain, modification, and loss of dimorphic pigmentation among Sophophora fruit flies. We show these CREs to be necessary components of a pigmentation GRN, as deletion from Drosophila melanogaster (derived dimorphic phenotype) resulted in lost expression and lost male‐specific pigmentation. We evaluated the ability of orthologous CRE sequences to drive reporter gene expression in species with modified (Drosophila auraria), secondarily lost (Drosophila ananassae), and ancestrally absent (Drosophila willistoni) pigmentation. We show that the transgene host frequently determines CRE activity, implicating trans‐evolution as a significant factor for this trait's diversity. We validated the gain of dimorphic Bab transcription factor expression as a trans‐change contributing to the dimorphic trait. Our findings suggest an amenability to change for the landscape of trans‐regulators and begs for an explanation as to why this is so common compared to the evolution of differentiation gene CREs. Research Highlights: cis‐regulatory elements (CREs) for Drosophila melanogaster tan and yellow genes drive dimorphic gene expression and pigmentation.Tests of CREs in four species reveal widespread trans‐regulatory evolution in a dimorphic gene regulatory network. [ABSTRACT FROM AUTHOR]

Skeletal development is tightly coordinated by chondrocytes and osteoblasts, which are derived from skeletal progenitors, and distinct cell-type gene regulatory programs underlie the specification and differentiation of cells. Runt-related transcription factor 2 (Runx2) is essential to chondrocyte hypertrophy and osteoblast differentiation. Genetic studies have revealed the biological functions of Runx2 and its involvement in skeletal genetic diseases. Meanwhile, molecular biology has provided a framework for our understanding of RUNX2-mediated transactivation at a limited number of cis-regulatory elements. Furthermore, studies using next-generation sequencing (NGS) have provided information on RUNX2-mediated gene regulation at the genome level and novel insights into the multiple layers of gene regulatory mechanisms, including the modes of action of RUNX2, chromatin accessibility, the concept of pioneer factors and phase separation, and three-dimensional chromatin organization. In this review, I summarize the emerging RUNX2-mediated regulatory mechanism from a multi-layer perspective and discuss future perspectives for applications in the treatment of skeletal diseases. [ABSTRACT FROM AUTHOR]

How complex morphological patterns form is an intriguing question in developmental biology. However, the mechanisms that generate complex patterns remain largely unknown. Here, we sought to identify the genetic mechanisms that regulate the tan (t) gene in a multi-spotted pigmentation pattern on the abdomen and wings of Drosophila guttifera. Previously, we showed that yellow (y) gene expression completely prefigures the abdominal and wing pigment patterns of this species. In the current study, we demonstrate that the t gene is co-expressed with the y gene in nearly identical patterns, both transcripts foreshadowing the adult abdominal and wing melanin spot patterns. We identified cis-regulatory modules (CRMs) of t, one of which drives reporter expression in six longitudinal rows of spots on the developing pupal abdomen, while the second CRM activates the reporter gene in a spotted wing pattern. Comparing the abdominal spot CRMs of y and t, we found a similar composition of putative transcription factor binding sites that are thought to regulate the complex expression patterns of both terminal pigmentation genes y and t. In contrast, the y and t wing spots appear to be regulated by distinct upstream factors. Our results suggest that the D. guttifera abdominal and wing melanin spot patterns have been established through the co-regulation of y and t, shedding light on how complex morphological traits may be regulated through the parallel coordination of downstream target genes. [ABSTRACT FROM AUTHOR]

Uncovering the cis -regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis -regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis -regulatory code. The cis -regulatory code dictates how DNA sequence controls quantitative transcription levels of each gene depending on cellular context. Kim and Wysocka review progress and challenges in understanding the layers of this code, from transcription factor binding to cofactor recruitment and ultimately cis -regulatory element specificity and function within complex regulatory landscapes. [ABSTRACT FROM AUTHOR]

Nucleosome-free open chromatin often harbors transcription factor (TF)-binding sites that are associated with active cis-regulatory elements. However, analysis of open chromatin regions has rarely been applied to oomycete or fungal plant pathogens. In this study, we performed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to identify open chromatin and cis-regulatory elements in Phytophthora sojae at the mycelial stage. We identified 10,389 peaks representing nucleosome-free regions (NFRs). The peaks were enriched in gene-promoter regions and associated with 40% of P. sojae genes; transcription levels were higher for genes with multiple peaks than genes with a single peak and were higher for genes with a single peak than genes without peak. Chromatin accessibility was positively correlated with gene transcription level. Through motif discovery based on NFR peaks in core promoter regions, 25 candidate cis-regulatory motifs with evidence of TF-binding footprints were identified. These motifs exhibited various preferences for location in the promoter region and associations with the transcription level of their target genes, which included some putative pathogenicity-related genes. As the first study revealing the landscape of open chromatin and the correlation between chromatin accessibility and gene transcription level in oomycetes, the results provide a technical reference and data resources for future studies on the regulatory mechanisms of gene transcription.[Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Summary: Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid–polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix–loop–helix (bHLH) transcription factors play a central role.By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐CRISPR associated protein 9 (CRISPR‐Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate‐inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis.Double myc1 myc2 loss‐of‐function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α‐tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR‐Cas9(VQR) genome editing of a specific cis‐regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility.Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress‐inducible specialized metabolism. [ABSTRACT FROM AUTHOR]

Cellular lineage determination is controlled by combinations of lineage-selective transcription factors (TFs) and associated coregulators that bind to cis -regulatory elements in DNA and regulate gene expression. The ability of these factors to regulate transcription is determined not only by their cooperativity, but also by biochemical and structural properties of the chromatin, sculpting higher-order genome organization. Here, we review recent advances in the understanding of the interplay between chromatin topology and transcription. Studies from many different fields, including adipocyte lineage determination, indicate that lineage determination and differentiation are dependent on elaborate crosstalk between cis -regulatory elements, leading to the formation of transcriptional hubs. Chromatin topology appears to provide a dynamic and supportive, rather than a deterministic, scaffold for this crosstalk. Chromatin, with all its associated regulatory proteins, intrinsically compartmentalizes to form transcription factor hubs and chromatin nanodomains. The overall chromatin scaffold, including topologically associating domain (TAD) structure and enhancer communities, is formed during the stem cell state. De novo activation of enhancers by lineage-selective TFs appears to be the main driver of the increased connectivity in lineage-selective sub-TADs and highly interconnected enhancer communities. Crosstalk between many different cis-regulatory elements via protein – protein interactions or by phase condensates has a key role in driving lineage determination and expression of cell identity genes. Master regulators act on a pre-established enhancer network to switch on cell identity genes. Mechanisms may involve de novo enhancer activation and/or increased activity of TF hubs/condensates. [ABSTRACT FROM AUTHOR]

Abstract Background The transcription of developmental regulatory genes is often controlled by multiple cis-regulatory elements. The identification and functional characterization of distal regulatory elements remains challenging, even in tractable model organisms like sea urchins. Results We evaluate the use of chromatin accessibility, transcription and RNA Polymerase II for their ability to predict enhancer activity of genomic regions in sea urchin embryos. ATAC-seq, PRO-seq, and Pol II ChIP-seq from early and late blastula embryos are manually contrasted with experimental cis-regulatory analyses available in sea urchin embryos, with particular attention to common developmental regulatory elements known to have enhancer and silencer functions differentially deployed among embryonic territories. Using the three functional genomic data types, machine learning models are trained and tested to classify and quantitatively predict the enhancer activity of several hundred genomic regions previously validated with reporter constructs in vivo. Conclusions Overall, chromatin accessibility and transcription have substantial power for predicting enhancer activity. For promoter-overlapping cis-regulatory elements in particular, the distribution of Pol II is the best predictor of enhancer activity in blastula embryos. Furthermore, ATAC- and PRO-seq predictive value is stage dependent for the promoter-overlapping subset. This suggests that the sequence of regulatory mechanisms leading to transcriptional activation have distinct relevance at different levels of the developmental gene regulatory hierarchy deployed during embryogenesis.

The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in most gnathostomes but undetectable in the jawless hagfish and lamprey. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 results in a significant reduction of early gene expression of nkx3.2 and leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

A newly discovered enhancer region may have allowed vertebrates to evolve the ability to open and close their jaws.

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50–75% ammonium–sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment.

The transcription factor WRINKLED1 (WRI1) is known as a master regulator of fatty acid synthesis in developing oilseeds of Arabidopsis thaliana and other species. WRI1 is known to directly stimulate the expression of many fatty acid biosynthetic enzymes and a few targets in the lower part of the glycolytic pathway. However, it remains unclear to what extent and how the conversion of sugars into fatty acid biosynthetic precursors is controlled by WRI1. To shortlist possible gene targets for future in-planta experimental validation, here we present a strategy that combines phylogenetic foot printing of cisregulatory elements with additional layers of evidence. Upstream regions of protein-encoding genes in A. thaliana were searched for the previously described DNA-binding consensus for WRI1, the ASML1/WRI1 (AW)-box. For about 900 genes, AW-box sites were found to be conserved across orthologous upstream regions in 11 related species of the crucifer family. For 145 select potential target genes identified this way, affinity of upstream AW-box sequences to WRI1 was assayed by Microscale Thermophoresis. This allowed definition of a refined WRI1 DNA-binding consensus. We find that known WRI1 gene targets are predictable with good confidence when upstream AW-sites are phylogenetically conserved, specifically binding WRI1 in the in vitro assay, positioned in proximity to the transcriptional start site, and if the gene is co-expressed with WRI1 during seed development. When targets predicted in this way are mapped to central metabolism, a conserved regulatory blueprint emerges that infers concerted control of contiguous pathway sections in glycolysis and fatty acid biosynthesis by WRI1. Several of the newly predicted targets are in the upper glycolysis pathway and the pentose phosphate pathway. Of these, plastidic isoforms of fructokinase (FRK3) and of phosphoglucose isomerase (PGI1) are particularly corroborated by previously reported seed phenotypes of respective null mutations. [ABSTRACT FROM AUTHOR]