Transcriptional Regulatory Dynamics Research Articles

Quantification and visualization of cis-regulatory dynamics in single-cell multi-omics data with TREASMO.

Recent advances in single-cell multi-omics technologies have provided unprecedented insights into regulatory processes. We introduce TREASMO, a versatile Python package designed to quantify and visualize transcriptional regulatory dynamics in single-cell multi-omics datasets. TREASMO has four modules, spanning data preparation, correlation quantification, downstream analysis and visualization, enabling comprehensive dataset exploration. By introducing a novel single-cell gene-peak correlation strength index, TREASMO facilitates accurate identification of regulatory changes at single-cell resolution. Validation on a hematopoietic stem and progenitor cell dataset showcases TREASMO's capacity in quantifying the gene-peak correlation strength at the single-cell level, identifying regulatory markers and discovering temporal regulatory patterns along the trajectory.

NRG1 Regulates Proliferation, Migration and Differentiation of Human Limbal Epithelial Stem Cells.

Limbal epithelial stem/progenitor cells (LESCs) proliferate, migrate and differentiate into mature corneal epithelium cells (CECs) that cover the ocular surface. LESCs play a crucial role in the maintenance and regeneration of the corneal epithelium, and their dysfunction can lead to various corneal diseases. Neuregulin 1 (NRG1) is a member of the epidermal growth factor family that regulates the growth and differentiation of epithelial tissues. Here, we depicted the dynamic transcriptomic profiles during human CEC differentiation, identifying six gene co-expression modules that were specific to different differentiation stages. We found that the expression of NRG1 was high in human LESCs and decreased dramatically upon differentiation. Knockdown of NRG1 significantly inhibited LESC proliferation and upregulated the expression of the terminal differentiation marker genes KRT3, KRT12 and CLU. In addition, the scratch wound closure assay showed that knockdown of NRG1 attenuated wound closure of LESCs over 24 h. Together, we dissected the transcriptional regulatory dynamics during CEC differentiation and identified NRG1 as a key regulator that promoted LESC proliferation and migration and maintained the undifferentiated state.

Temporal and regulatory dynamics of the inner ear transcriptome during development in mice

The inner ear controls hearing and balance, while the temporal molecular signatures and transcriptional regulatory dynamics underlying its development are still unclear. In this study, we investigated time-series transcriptome in the mouse inner ear from embryonic day 11.5 (E11.5) to postnatal day 7 (P7) using bulk RNA-Seq. A total of 10,822 differentially expressed genes were identified between pairwise stages. We identified nine significant temporal expression profiles using time-series expression analysis. The constantly down-regulated profiles throughout the development are related to DNA activity and neurosensory development, while the constantly upregulated profiles are related to collagen and extracellular matrix. Further co-expression network analysis revealed that several hub genes, such as Pnoc, Cd9, and Krt27, are related to the neurosensory development, cell adhesion, and keratinization. We uncovered three important transcription regulatory paths during mice inner ear development. Transcription factors related to Hippo/TGFβ signaling induced decreased expressions of genes related to the neurosensory and inner ear development, while a series of INF genes activated the expressions of genes in immunoregulation. In addition to deepening our understanding of the temporal and regulatory mechanisms of inner ear development, our transcriptomic data could fuel future multi-species comparative studies and elucidate the evolutionary trajectory of auditory development.

Dynamic chromatin state profiling reveals regulatory roles of auxin and cytokinin in shoot regeneration.

Shoot regeneration is mediated by the sequential action of two phytohormones, auxin and cytokinin. However, the chromatin regulatory landscapes underlying this dynamic response have not yet been studied. In this study, we jointly profiled chromatin accessibility, histone modifications, and transcriptomes to demonstrate that a high auxin/cytokinin ratio environment primes Arabidopsis shoot regeneration by increasing the accessibility of the gene loci associated with pluripotency and shoot fate determination. Cytokinin signaling not only triggers the commitment of the shoot progenitor at later stages but also allows chromatin to maintain shoot identity genes at the priming stage. Our analysis of transcriptional regulatory dynamics further identifies a catalog of regeneration cis-elements dedicated to cell fate transitions and uncovers important roles of BES1, MYC, IDD, and PIF transcription factors in shoot regeneration. Our results, thus, provide a comprehensive resource for studying cell reprogramming in plants and provide potential targets for improving future shoot regeneration efficiency.

Dynamic Chromatin State Profiling Reveals Regulatory Principle of Pluripotency During Shoot Regeneration

Genome-editing technologies have advanced in recent years but designing future crops is still limited by current methods of improving shoot regenerative capacity. Shoot regeneration is mediated by the sequential action of two phytohormones, auxin and cytokinin. However, the chromatin regulatory landscapes underlying this dynamic response have not yet been studied. In this study, we jointly profiled chromatin accessibility, histone modifications, and transcriptomes to demonstrate that a high-auxin environment primes shoot regeneration by increasing the accessibility of the gene loci associated with pluripotency and shoot fate determination. Cytokinin signaling not only triggers the commitment of the shoot progenitor at later stages, but allows chromatin to maintain shoot identity genes at the priming stage. Our analysis of transcriptional regulatory dynamics further identifies a catalog of regeneration cis-elements dedicated to cell fate transitions, and uncovers previously unrecognized roles of BES1, MYC, IDD, and PIF transcription factors in shoot regeneration. Our results provide a comprehensive resource for studying somatic cell reprogramming in plants and provides potential targets for improving future shoot regeneration efficiency.

Quantitative dissection of transcription in development yields evidence for transcription-factor-driven chromatin accessibility

Thermodynamic models of gene regulation can predict transcriptional regulation in bacteria, but in eukaryotes, chromatin accessibility and energy expenditure may call for a different framework. Here, we systematically tested the predictive power of models of DNA accessibility based on the Monod-Wyman-Changeux (MWC) model of allostery, which posits that chromatin fluctuates between accessible and inaccessible states. We dissected the regulatory dynamics of hunchback by the activator Bicoid and the pioneer-like transcription factor Zelda in living Drosophila embryos and showed that no thermodynamic or non-equilibrium MWC model can recapitulate hunchback transcription. Therefore, we explored a model where DNA accessibility is not the result of thermal fluctuations but is catalyzed by Bicoid and Zelda, possibly through histone acetylation, and found that this model can predict hunchback dynamics. Thus, our theory-experiment dialogue uncovered potential molecular mechanisms of transcriptional regulatory dynamics, a key step toward reaching a predictive understanding of developmental decision-making.

Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors.

Vertebrate appendage regeneration requires precisely coordinated remodeling of the transcriptional landscape to enable the growth and differentiation of new tissue, a process executed over multiple days and across dozens of cell types. The heterogeneity of tissues and temporally-sensitive fate decisions involved has made it difficult to articulate the gene regulatory programs enabling regeneration of individual cell types. To better understand how a regenerative program is fulfilled by neural progenitor cells (NPCs) of the spinal cord, we analyzed pax6-expressing NPCs isolated from regenerating Xenopus tropicalis tails. By intersecting chromatin accessibility data with single-cell transcriptomics, we find that NPCs place an early priority on neuronal differentiation. Late in regeneration, the priority returns to proliferation. Our analyses identify Pbx3 and Meis1 as critical regulators of tail regeneration and axon organization. Overall, we use transcriptional regulatory dynamics to present a new model for cell fate decisions and their regulators in NPCs during regeneration.

Genome and transcriptome evolve separately in recently hybridized Trichosporon fungi

Genome hybridization is an important evolutionary event that gives rise to species with novel capabilities. However, the merging of distinct genomes also brings together incompatible regulatory networks that must be resolved during the course of evolution. Understanding of the early stages of post-hybridization evolution is particularly important because changes in these stages have long-term evolutionary consequences. Here, via comparative transcriptomic analyses of two closely related, recently hybridized Trichosporon fungi, T. coremiiforme and T. ovoides, and three extant relatives, we show that early post-hybridization evolutionary processes occur separately at the gene sequence and gene expression levels but together contribute to the stabilization of hybrid genome and transcriptome. Our findings also highlight lineage-specific consequences of genome hybridization, revealing that the transcriptional regulatory dynamics in these hybrids responded completely differently to gene loss events: one involving both subgenomes and another that is strictly subgenome-specific.

Charting the transcriptional regulatory changes in mouse liver during fasting.

Summary In a recent paper: Goldstein I, Baek S, Presman DM, Paakinaho V, Swinstead EE, Hager GL. Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response. Genome Res. 2017 Mar;27(3):427-39., the authors used a number of functional genomics approaches to explore the transcriptional regulatory dynamics that occur during hepatic fasting. They used chromatin landscape data to identify key fasting-related transcription factors, four of which were further investigated because they are known players of the fasting response: CEBPB (CCAAT enhancer binding-beta), CREB1 (cAMP responsive element binding protein I), GR (glucocorticoid receptor), and PPARA (peroxisome proliferator activated receptor alpha). The authors described two operating modules (GR-CREB1 and PPARA-GR) with synergistic effects driving gluconeogenesis through an assisted loading model and fatty acid oxidation/ketogenesis through a transcription factor cascade, respectively. Finally, using single-cell tracking, they confirmed that GR facilitates CREB1 binding to DNA. This article is protected by copyright. All rights reserved.

Differential co-expression analysis reveals a novel prognostic gene module in ovarian cancer

Ovarian cancer is one of the most significant disease among gynecological disorders that women suffered from over the centuries. However, disease-specific and effective biomarkers were still not available, since studies have focused on individual genes associated with ovarian cancer, ignoring the interactions and associations among the gene products. Here, ovarian cancer differential co-expression networks were reconstructed via meta-analysis of gene expression data and co-expressed gene modules were identified in epithelial cells from ovarian tumor and healthy ovarian surface epithelial samples to propose ovarian cancer associated genes and their interactions. We propose a novel, highly interconnected, differentially co-expressed, and co-regulated gene module in ovarian cancer consisting of 84 prognostic genes. Furthermore, the specificity of the module to ovarian cancer was shown through analyses of datasets in nine other cancers. These observations underscore the importance of transcriptome based systems biomarkers research in deciphering the elusive pathophysiology of ovarian cancer, and here, we present reciprocal interplay between candidate ovarian cancer genes and their transcriptional regulatory dynamics. The corresponding gene module might provide new insights on ovarian cancer prognosis and treatment strategies that continue to place a significant burden on global health.

Transcriptional regulatory dynamics drive coordinated metabolic and neural response to social challenge in mice.

Agonistic encounters are powerful effectors of future behavior, and the ability to learn from this type of social challenge is an essential adaptive trait. We recently identified a conserved transcriptional program defining the response to social challenge across animal species, highly enriched in transcription factor (TF), energy metabolism, and developmental signaling genes. To understand the trajectory of this program and to uncover the most important regulatory influences controlling this response, we integrated gene expression data with the chromatin landscape in the hypothalamus, frontal cortex, and amygdala of socially challenged mice over time. The expression data revealed a complex spatiotemporal patterning of events starting with neural signaling molecules in the frontal cortex and ending in the modulation of developmental factors in the amygdala and hypothalamus, underpinned by a systems-wide shift in expression of energy metabolism-related genes. The transcriptional signals were correlated with significant shifts in chromatin accessibility and a network of challenge-associated TFs. Among these, the conserved metabolic and developmental regulator ESRRA was highlighted for an especially early and important regulatory role. Cell-type deconvolution analysis attributed the differential metabolic and developmental signals in this social context primarily to oligodendrocytes and neurons, respectively, and we show that ESRRA is expressed in both cell types. Localizing ESRRA binding sites in cortical chromatin, we show that this nuclear receptor binds both differentially expressed energy-related and neurodevelopmental TF genes. These data link metabolic and neurodevelopmental signaling to social challenge, and identify key regulatory drivers of this process with unprecedented tissue and temporal resolution.

Transcriptional regulatory dynamics of the hypothalamic-pituitary-testicular axis in male mice exposed to fluoride

Transcriptional regulatory dynamics of the hypothalamic-pituitary-testicular axis in male mice exposed to fluoride

Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics.

Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

"Hit-and-run": Transcription factors get caught in the act.

A key challenge for understanding transcriptional regulation is being able to measure transcription factor (TF)-DNA binding events with sufficient spatial and temporal resolution; that is, when and where TFs occupy their cognate sites. A recent study by Para et al. has highlighted the dynamics underlying the activation of gene expression by a master regulator TF. This study provides concrete evidence for a long-standing hypothesis in biology, the "hit-and-run" mechanism, which was first proposed decades ago. That is, gene expression is dynamically controlled by a TF that transiently binds and activates a target gene, which might stay in a transcriptionally active state after the initial binding event has ended. Importantly, the experimental procedure introduced, TARGET, provides a useful way for identifying multiple target genes transiently bound by their regulators, which can be used in conjunction with other well-established methods to improve our understanding of transcriptional regulatory dynamics.

C/EBPα and C/EBPβ mediated genome‐wide combinatorial transcriptional regulatory dynamics during early onset of liver regeneration and chronic alcohol intake (398.6)

The primary objective of this study is to characterize effects of chronic alcohol consumption on genome‐scale transcriptional regulatory network dynamics during the early phase liver regeneration after 70% partial hepatectomy (PHx) at 6h post 2/3rd PHx in the rat. Rats were fed liquid diet containing 36% of total calories derived from ethanol for 5 weeks, controls were pair‐fed an isocaloric liquid diet with carbohydrate replacing ethanol. Genome‐wide binding targets C/EBPα and C/EBPβ were detected by ABI SOLiD sequencing approach and qPCR validation.Our results reveal C/EBPα binding to promoters of metabolic regulator genes, cell cycle inhibitors, glucose transporter, dehydrogenase and reductase family member, RNA metabolic, phosphate metabolic genes, etc. C/EBPβ was detected at the gene promoters of key regulatory pathways such as regulation of cell cycle, regulation of programmed cell death, regulation of cAMP biosynthetic process, positive regulation of RNA metabolic process, MAPK signaling, Insulin signaling, cell adhesion, etc. We performed qPCR validation of a set of genomic loci with increase in C/EBPα and C/EBPβ activity in the ethanol versus control group. We used our PAINT and TRANSFAC bioinformatics software to predict combinatorial regulatory modules containing co‐localized binding sites for C/EBPα and C/EBPβ, on key genes promoter regions. Support: K05 AA017261, and T32 AA007463.

Genome‐wide combinatorial transcriptional regulatory dynamics during early onset of liver regeneration and chronic alcohol intake

The objective of this study is to identify genome‐scale transcriptional regulatory network dynamics affected by chronic alcohol intake during the early phase of liver regeneration. Rats were fed liquid diet containing 36% of total calories derived from ethanol for 5 weeks, and controls were pair‐fed an isocaloric liquid diet. A 70% partial hepatectomy (PHx) was performed. Genome‐wide binding targets for NF‐kB, C/EBP‐beta, and STAT3 were detected either by Roche NimbleGen ChIP‐chip or ABI SOLiD sequencing approach. Our findings indicate significant differences in the binding profiles of all the three regulators in chronic ethanol compared to isocaloric controls, at baseline as well as in response to PHx. Pathway analysis revealed that potential target genes specific to the chronic ethanol group participate in key processes such as mitochondria processing, DNA methylation, cell death, proteolysis, histone modification, lipid biosynthetic process, and regulation of cell cycle etc. Our results revealed combinatorial binding dynamics of multiple TFs from ChIP‐seq and DNA binding motif. Our results provide a first system‐wide view of the regulatory network dynamics in liver regeneration as it is affected by alcohol, and implicate combinatorial regulatory modules as participating in the adverse liver regeneration response. Research Support: NIH R01 AA018873, K05 AA017261, and T32 AA007463.

Genome‐wide combinatorial transcriptional regulatory dynamics during early onset of liver regeneration and chronic alcohol intake

The objective of this study is to characterize effects of chronic alcohol consumption on genome‐scale transcriptional regulatory network dynamics during the early phase liver regeneration after 70% partial hepatectomy (PHx) at 6h post 2/3rd PHx in the rat. Rats were fed liquid diet containing 36% of total calories derived from ethanol for 5 weeks, controls were pair‐fed an isocaloric liquid diet with carbohydrate replacing ethanol. Genome‐wide binding targets for NF‐kB, C/EBPbeta, and STAT3 were detected either by Roche NimbleGen ChIP‐chip or ABI SOLID sequencing approach. Our results indicate significant differences in the binding profiles of all the three regulators in chronic ethanol compared to isocaloric controls, at baseline as well as in response to PHx. Pathway analysis revealed that potential target genes specific to the chronic ethanol group participate in key processes such as mitochondria processing, DNA methylation, cell death, proteolysis, histone modification, lipid biosynthetic process, and regulation of cell cycle. Our results provide a first system‐wide view of the regulatory network dynamics in liver regeneration as it is affected by alcohol, and implicate combinatorial regulatory modules as participating in the adverse liver regeneration response. Research Support: NIH AA018873 and AA017261.

Transcriptional regulatory dynamics of the hypothalamic–pituitary–gonadal axis and its peripheral pathways as impacted by the 3-beta HSD inhibitor trilostane in zebrafish ( Danio rerio)

To study mechanisms underlying generalized effects of 3β hydroxysteroid dehydrogenase (HSD3B) inhibition, reproductively mature zebrafish ( Danio rerio) were exposed to trilostane at two dosages for 24, 48, or 96 h and their gonadal RNA samples profiled with Agilent zebrafish microarrays. Trilostane had substantial impact on the transcriptional dynamics of zebrafish, as reflected by a number of differentially expressed genes (DEGs) including transcription factors (TFs), altered TF networks, signaling pathways, and Gene Ontology (GO) biological processes. Changes in gene expression between a treatment and its control were mostly moderate, ranging from 1.3 to 2.0 fold. Expression of genes coding for HSD3B and many of its transcriptional regulators remained unchanged, suggesting transcriptional up-regulation is not a primary compensatory mechanism for HSD3B enzyme inhibition. While some trilostane-responsive TFs appear to share cellular functions linked to endocrine disruption, there are also many other DEGs not directly linked to steroidogenesis. Of the 65 significant TF networks, little similarity, and therefore little cross-talk, existed between them and the hypothalamic–pituitary–gonadal (HPG) axis. The most enriched GO biological processes are regulations of transcription, phosphorylation, and protein kinase activity. Most of the impacted TFs and TF networks are involved in cellular proliferation, differentiation, migration, and apoptosis. While these functions are fairly broad, their underlying TF networks may be useful to development of generalized toxicological screening methods. These findings suggest that trilostane-induced effects on fish endocrine functions are not confined to the HPG-axis alone. Its impact on corticosteroid synthesis could also have contributed to some system wide transcriptional changes in zebrafish observed in this study.