scholarly journals First responders shape a prompt and sharp NF-κB–mediated transcriptional response to TNF-α

2020 ◽  
Author(s):  
Samuel Zambrano ◽  
Alessia Loffreda ◽  
Elena Carelli ◽  
Giacomo Stefanelli ◽  
Federica Colombo ◽  
...  
Keyword(s):  
Target Genes ◽  
Single Cells ◽  
Gene Activation ◽  
Transcriptional Response ◽  
First Responders ◽  
Population Level ◽  
Transcription Activator ◽  
Transcriptional Responses ◽  
Time Resolved ◽  
Refractory State

SummaryNF-κB acts as the master regulator of the transcriptional response to inflammatory signals by translocating into the nucleus upon stimuli, but we lack a single-cell characterization of the resulting transcription dynamics. Here we show that transcription of NF-κB target genes is strongly heterogeneous in individual cells but dynamically coordinated at the population level, since the average nascent transcription is prompt (i.e. occurs almost immediately) and sharp (i.e. increases and decreases rapidly) compared to NF-κB nuclear localization. Using an NF-κB-controlled MS2 reporter we confirm that the population-level transcriptional activity emerges from a strongly heterogeneous response in single cells as compared to NF-κB translocation dynamics, including the presence of a fraction of “first responders”. Mathematical models show that a combination of NF-κB mediated gene activation and a gene activity module including a gene refractory state is enough to produce sharp and prompt transcriptional responses. Our data and models show how the expression of the target genes of a paradigmatic inducible transcription activator upon stimuli can be time-resolved at population level and yet heterogeneous across single cells.

scholarly journals Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

2018 ◽  
Author(s):  
Onur Tidin ◽  
Elias T. Friman ◽  
Felix Naef ◽  
David M. Suter
Keyword(s):  
Transcriptional Activation ◽  
Target Gene ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Tissue Growth ◽  
Live Cells ◽  
High Temporal Resolution ◽  
Transcriptional Responses

AbstractThe transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-β stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.

scholarly journals Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

2013 ◽  
Vol 368 (1632) ◽  
pp. 20130018 ◽  
Author(s):  
Andrea I. Ramos ◽  
Scott Barolo
Keyword(s):  
Transcription Factor ◽  
Binding Affinity ◽  
Binding Sites ◽  
Target Genes ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Morphogen Gradients ◽  
Weak Binding ◽  
Initial Hypothesis

In the era of functional genomics, the role of transcription factor (TF)–DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients . We noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp , wingless and stripe , by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.

scholarly journals Induction of Mxi1-SRα by FOXO3a Contributes to Repression of Myc-Dependent Gene Expression

2007 ◽  
Vol 27 (13) ◽  
pp. 4917-4930 ◽  
Author(s):  
Oona Delpuech ◽  
Beatrice Griffiths ◽  
Philip East ◽  
Abdelkader Essafi ◽  
Eric W.-F. Lam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle Progression ◽  
Dna Microarrays ◽  
Target Genes ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Inhibitory Effect ◽  
Pi3 Kinase ◽  
Promoter Regions ◽  
Inhibition Of Proliferation

ABSTRACT Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. FOXOs have been implicated in the regulation of cell cycle progression, oxidative stress resistance, and apoptosis. Using DNA microarrays, we analyzed the transcriptional response to FOXO3a activation by gene expression analysis in DLD-1 colon cancer cells stably expressing a FOXO3a.A3-ER fusion protein. We found that activation of FOXO3a resulted in repression of a number of previously identified Myc target genes. Furthermore, FOXO3a activation induced expression of several members of the Mad/Mxd family of transcriptional repressors, most notably Mxi1. The induction of Mxi1 by FOXO3a was specific to the Mxi1-SRα isoform and was mediated by three highly conserved FOXO binding sites within the first intron of the gene. Activation of FOXO3a in response to inhibition of Akt also resulted in activation of Mxi1-SRα expression. Silencing of Mxi1 by small interfering RNA (siRNA) reduced FOXO3a-mediated repression of a number of Myc target genes. We also observed that FOXO3a activation induced a switch in promoter occupancy from Myc to Mxi1 on the E-box containing promoter regions of two Myc target genes, APEX and FOXM1. siRNA-mediated transient silencing of Mxi1 or all Mad/Mxd proteins reduced exit from S phase in response to FOXO3a activation, and stable silencing of Mxi1 or Mad1 reduced the growth inhibitory effect of FOXO3a. We conclude that induction of Mad/Mxd proteins contributes to the inhibition of proliferation in response to FOXO3a activation. Our results provide evidence of direct regulation of Mxi1 by FOXO3a and imply an additional mechanism through which the PI3-kinase/Akt/FOXO pathway can modulate Myc function.

scholarly journals MON-727 The Chromatin Landscape of Glucocorticoid Regulated Genes in Mouse Embryonic Neural Stem / Progenitor Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Kimberly Berry ◽  
Uma Chandran ◽  
Fangping Mu ◽  
Donald DeFranco
Keyword(s):  
Progenitor Cells ◽  
Target Genes ◽  
Therapeutic Potential ◽  
Transcriptional Response ◽  
Clinical Intervention ◽  
Enrichment Analysis ◽  
Open Chromatin ◽  
Transcriptional Responses ◽  
Embryonic Mouse

Abstract Stem/Progenitor Cells Antenatal administration of Dexamethasone (Dex), a synthetic glucocorticoid (GC), is a common clinical intervention for women at risk for preterm birth or in preterm labor that effectively reduces fetal risk of mortality and bronchopulmonary-related comorbidities. Despite the therapeutic potential of Dex, excess GC act adversely in the developing central nervous system to reprogram distinct neural circuits in the brain by acting through the glucocorticoid receptor (GR). For example, prenatal exposure to excess GCs can impact neural stem and progenitor cell (NSPC) proliferation leading to long-term alterations in prefrontal cortical neuronal complexity, which could contribute to behavioral and cognitive impairments later in life. The GR is a member of the nuclear receptor superfamily that, when bound by a ligand, translocates from the cytoplasm to the nucleus and associates indirectly or directly with DNA elements (e.g. glucocorticoid responsive elements or GREs) resulting in the activation and/or repression of target genes. While GR-regulated transcriptomes have been identified in many NSPC models, the mechanisms responsible for programming these cells for GC-responsiveness remain largely unknown. We therefore used transposase accessible chromatin followed by genome-wide sequencing (Omni ATAC-seq) to characterize the chromatin landscape of primary embryonic mouse NSPCs in response to an acute in vitro treatment with Dex. We identified a small, yet distinct fraction (0.002%, p<0.05) of open chromatin sites that were Dex-inducible. 95% of these Dex-induced changes in chromatin accessibility occur within intronic or intergenic regions, suggesting the presence of long-range enhancer-promoter contacts that mediate NSPC transcriptional responses to Dex. Motif enrichment analysis revealed putative GRE sites located in Dex-inducible open chromatin within -5kb/+2kb of a Dex-induced gene, providing possible DNA targets of GR for further validation. A number of other transcription factors implicated in neurodevelopmental processes were found to underlie both Dex-inducible and constitutively open chromatin regions. Characterization of the precise epigenetic and transcriptional response to excess GC in-utero, and its influence on acute and chronic neurological outcomes, will encourage the development of alternative GC treatment regimens that could protect the developing brain from insult while providing optimal health outcomes in neonates.

scholarly journals A Role for Iron-Sulfur Clusters in the Regulation of Transcription Factor Yap5-dependent High Iron Transcriptional Responses in Yeast

2012 ◽  
Vol 287 (42) ◽  
pp. 35709-35721 ◽  
Author(s):  
Liangtao Li ◽  
Ren Miao ◽  
Sophie Bertram ◽  
Xuan Jia ◽  
Diane M. Ward ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Transcriptional Response ◽  
Regulation Of Transcription ◽  
Transcriptional Responses ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Sulfur Cluster ◽  
High Iron ◽  
Iron Sulfur

Yeast respond to increased cytosolic iron by activating the transcription factor Yap5 increasing transcription of CCC1, which encodes a vacuolar iron importer. Using a genetic screen to identify genes involved in Yap5 iron sensing, we discovered that a mutation in SSQ1, which encodes a mitochondrial chaperone involved in iron-sulfur cluster synthesis, prevented expression of Yap5 target genes. We demonstrated that mutation or reduced expression of other genes involved in mitochondrial iron-sulfur cluster synthesis (YFH1, ISU1) prevented induction of the Yap5 response. We took advantage of the iron-dependent catalytic activity of Pseudaminobacter salicylatoxidans gentisate 1,2-dioxygenase expressed in yeast to measure changes in cytosolic iron. We determined that reductions in iron-sulfur cluster synthesis did not affect the activity of cytosolic gentisate 1,2-dioxygenase. We show that loss of activity of the cytosolic iron-sulfur cluster assembly complex proteins or deletion of cytosolic glutaredoxins did not reduce expression of Yap5 target genes. These results suggest that the high iron transcriptional response, as well as the low iron transcriptional response, senses iron-sulfur clusters.

scholarly journals Single-cell RNA-seq reveals CD16- monocytes as key regulators of human monocyte transcriptional response to Toxoplasma

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Anirudh Patir ◽  
Anton Gossner ◽  
Prakash Ramachandran ◽  
Joana Alves ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Peripheral Blood ◽  
Control Strategies ◽  
Transcriptional Response ◽  
Population Level ◽  
Human Monocyte ◽  
Cell Populations ◽  
Transcriptional Responses ◽  
Average Cell ◽  
First Time

AbstractMonocytes are among the major myeloid cells that respond to Toxoplasma, a ubiquitous foodborne that infects ≥ 1 billion people worldwide, in human peripheral blood. As such, a molecular understanding of human monocyte-Toxoplasma interactions can expedite the development of novel human toxoplasmosis control strategies. Current molecular studies on monocyte-Toxoplasma interactions are based on average cell or parasite responses across bulk cell populations. Although informative, population-level averages of monocyte responses to Toxoplasma have sometimes produced contradictory results, such as whether CCL2 or IL12 define effective monocyte responses to the parasite. Here, we used single-cell dual RNA sequencing (scDual-Seq) to comprehensively define, for the first time, the monocyte and parasite transcriptional responses that underpin human monocyte-Toxoplasma encounters at the single cell level. We report extreme transcriptional variability between individual monocytes. Furthermore, we report that Toxoplasma-exposed and unexposed monocytes are transcriptionally distinguished by a reactive subset of CD14+CD16- monocytes. Functional cytokine assays on sorted monocyte populations show that the infection-distinguishing monocytes secrete high levels of chemokines, such as CCL2 and CXCL5. These findings uncover the Toxoplasma-induced monocyte transcriptional heterogeneity and shed new light on the cell populations that largely define cytokine and chemokine secretion in human monocytes exposed to Toxoplasma.

scholarly journals Permissive epigenetic landscape facilitates distinct transcriptional signatures of activating transcription factor 6 in the liver

2021 ◽  
Author(s):  
Anjana Ramdas Nair ◽  
Priyanka Lakhiani ◽  
Chi Zhang ◽  
Filippo Macchi ◽  
Kirsten C. Sadler
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Expression Patterns ◽  
Transcriptional Response ◽  
Differentially Expressed ◽  
Small Subset ◽  
Gene Promoters ◽  
Transcriptional Responses ◽  
Functional Studies ◽  
Activating Transcription Factor

ABSTRACTProteostatic stress initiates a transcriptional response that is unique to the stress condition, yet the regulatory mechanisms underlying the distinct gene expression patterns observed in stressed cells remains unknown. Using a functional genomic approach, we investigated how activating transcription factor 6 (ATF6), a key transcription factor in the unfolded protein response (UPR), regulates target genes. We first designed a computational strategy to define Atf6 target genes based on the evolutionary conservation of predicted ATF6 binding in gene promoters, identifying 652 conserved putative Atf6 target (CPAT) genes. CPATs were overrepresented for genes functioning in the UPR, however, the majority functioned in cellular processes unrelated to proteostasis, including small molecule metabolism and development. Functional studies of stress-independent and toxicant based Atf6 activation in zebrafish livers showed that the pattern of CPAT expression in response to Atf6 overexpression, alcohol and arsenic was unique. Only 34 CPATs were differentially expressed in all conditions, indicating that Atf6 is sufficient to regulate a small subset of CPATs. Blocking Atf6 using Ceapins in zebrafish demonstrated that Atf6 is necessary for activation of these genes in response to arsenic. We investigated CPAT during physiologically mediated hepatocyte stress using liver regeneration in mice as a model. Over half of all CPATs were differentially expressed during this process. This was attributed to the permissive chromatin environment in quiescent livers on the promoters of these genes, characterized by the absence of H3K27me3 and enrichment of H3K4me3. Taken together, these data uncover a complex transcriptional response to Atf6 activation and implicate a permissive epigenome as a mechanism by which distinct transcriptional responses are regulated by Atf6.

scholarly journals Hypoxia-inducible factor 3 biology: complexities and emerging themes

2016 ◽  
Vol 310 (4) ◽  
pp. C260-C269 ◽  
Author(s):  
Cunming Duan
Keyword(s):  
Transcription Initiation ◽  
Target Genes ◽  
Developmental Stages ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Response ◽  
Dominant Negative ◽  
Transcription Activator ◽  
Α Subunit ◽  
Naturally Occurring ◽  
Protein Functions

The hypoxia-inducible factor (HIF) family has three distinct members in most vertebrates. All three HIFs consist of a unique and oxygen-labile α-subunit and a common and stable β-subunit. While HIF-1 and HIF-2 function as master regulators of the transcriptional response to hypoxia, much less is known about HIF-3. The HIF-3α gene gives rise to multiple HIF-3α variants due to the utilization of different promoters, different transcription initiation sites, and alternative splicing. These HIF-3α variants are expressed in different tissues, at different developmental stages, and are differentially regulated by hypoxia and other factors. Recent studies suggest that different HIF-3α variants have different and even opposite functions. There is strong evidence that full-length HIF-3α protein functions as an oxygen-regulated transcription activator and that it activates a unique transcriptional program in response to hypoxia. Many HIF-3α target genes have been identified. While some short HIF-3α variants act as dominant-negative regulators of HIF-1/2α actions, other HIF-3α variants can inhibit HIF-1/2α actions by competing for the common HIF-β. There are also a number of HIF-3α variants yet to be explored. Future studies of these naturally occurring HIF-3α variants will provide new and important insights into HIF biology and may lead to the development of new therapeutic strategies.

scholarly journals Single-cell RNA-seq reveals CD16- monocytes as key regulators of human monocyte transcriptional response to Toxoplasma

2019 ◽  
Author(s):  
Anirudh Patir ◽  
Anton Gossner ◽  
Prakash Ramachandran ◽  
Joana Alves ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Peripheral Blood ◽  
Control Strategies ◽  
Transcriptional Response ◽  
Population Level ◽  
Human Monocyte ◽  
Cell Populations ◽  
Transcriptional Responses ◽  
Average Cell ◽  
First Time

ABSTRACTMonocytes are among the major myeloid cells that respond to Toxoplasma, a ubiquitous foodborne that infects ≥1 billion people worldwide, in human peripheral blood. As such, a molecular understanding of human monocyte-Toxoplasma interactions can expedite the development of novel human toxoplasmosis control strategies. Current molecular studies on monocyte-Toxoplasma interactions are based on average cell or parasite responses across bulk cell populations. Although informative, population-level averages of monocyte responses to Toxoplasma have sometimes produced contradictory results, such as whether CCL2 or IL12 define effective monocyte response to the parasite. Here, we used single-cell dual RNA sequencing (scDual-Seq) to comprehensively define, for the first time, the monocyte and parasite transcriptional responses that underpin human monocyte-Toxoplasma encounters at the single cell level. We report extreme transcriptional variability between individual monocytes. Furthermore, we report that Toxoplasma-exposed and unexposed monocytes are transcriptionally distinguished by a reactive subset of CD14++CD16- monocytes. Functional cytokine assays on sorted monocyte populations show that the infection-distinguishing monocytes secrete high levels of chemokines, such as CCL2 and CXCL5. These findings uncover the Toxoplasma-induced monocyte transcriptional heterogeneity and shed new light on the cell populations that largely define cytokine and chemokine secretion in human monocytes exposed to Toxoplasma.

scholarly journals TUP1, CPH1 and EFG1 Make Independent Contributions to Filamentation in Candida albicans

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Burkhard R Braun ◽  
Alexander D Johnson
Keyword(s):  
Candida Albicans ◽  
Environmental Conditions ◽  
Target Genes ◽  
Single Cells ◽  
Filamentous Growth ◽  
Surface Proteins ◽  
Pathway Expression ◽  
Separate Pathway ◽  
The Common ◽  
Growth Pathway

Abstract The common fungal pathogen, Candida albicans, can grow either as single cells or as filaments (hyphae), depending on environmental conditions. Several transcriptional regulators have been identified as having key roles in controlling filamentous growth, including the products of the TUP1, CPH1, and EFG1 genes. We show, through a set of single, double, and triple mutants, that these genes act in an additive fashion to control filamentous growth, suggesting that each gene represents a separate pathway of control. We also show that environmentally induced filamentous growth can occur even in the absence of all three of these genes, providing evidence for a fourth regulatory pathway. Expression of a collection of structural genes associated with filamentous growth, including HYR1, ECE1, HWP1, ALS1, and CHS2, was monitored in strains lacking each combination of TUP1, EFG1, and CPH1. Different patterns of expression were observed among these target genes, supporting the hypothesis that these three regulatory proteins engage in a network of individual connections to downstream genes and arguing against a model whereby the target genes are regulated through a central filamentous growth pathway. The results suggest the existence of several distinct types of filamentous forms of C. albicans, each dependent on a particular set of environmental conditions and each expressing a unique set of surface proteins.

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