scholarly journals Quantitative analysis of the ThrbCRM1-centered gene regulatory network

2019 ◽  
Author(s):  
Benjamin Souferi ◽  
Mark M. Emerson
Keyword(s):  
Transcription Factors ◽  
Regulatory Element ◽  
Critical Parameters ◽  
Reporter Plasmid ◽  
Response Curves ◽  
Enhancer Activity ◽  
Sequence Elements ◽  
Summary Statement ◽  
Specific Sequences

AbstractEnhancer activity is determined by both the activity and occupancy of transcription factors as well as the specific sequences they bind. Experimental investigation of this dynamic requires the ability to manipulate components of the system, ideally in as close to an in vivo context as possible. Here we use electroporation of plasmid reporters to define critical parameters of a specific cis-regulatory element, ThrbCRM1, during retinal development. ThrbCRM1 is associated with cone photoreceptor genesis and activated in a subset of developing retinal cells that co-express the Otx2 and Onecut1 (OC1) transcription factors. Variation of reporter plasmid concentration was used to generate dose response curves and revealed an effect of binding site availability on the number and strength of cells with reporter activity. Critical sequence elements of the ThrbCRM1 element were defined using both mutagenesis and misexpression of the Otx2 and OC1 transcription factors in the developing retina. Additionally, these experiments suggest that the ThrbCRM1 element is co-regulated by Otx2 and OC1 even under conditions of sub-optimal binding of OC1.Summary StatementSystematic variation of the levels of a transcriptional reporter plasmid, its trans-acting factors, and transcription factor binding sites reveals properties of a retinal enhancer during development.

Differences in the chromatin structure and cis-element organization of the human and mouse GATA1 loci: implications for cis-element identification

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3106-3116 ◽  
Author(s):  
Veronica Valverde-Garduno ◽  
Boris Guyot ◽  
Eduardo Anguita ◽  
Isla Hamlett ◽  
Catherine Porcher ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Sequence Comparison ◽  
Regulatory Element ◽  
Cis Elements ◽  
Enhancer Activity ◽  
Cis Element ◽  
Human Sequence ◽  
Human And Mouse

Abstract Cis-element identification is a prerequisite to understand transcriptional regulation of gene loci. From analysis of a limited number of conserved gene loci, sequence comparison has proved a robust and efficient way to locate cis-elements. Human and mouse GATA1 genes encode a critical hematopoietic transcription factor conserved in expression and function. Proper control of GATA1 transcription is critical in regulating myeloid lineage specification and maturation. Here, we compared sequence and systematically mapped position of DNase I hypersensitive sites, acetylation status of histone H3/H4, and in vivo binding of transcription factors over approximately 120 kilobases flanking the human GATA1 gene and the corresponding region in mice. Despite lying in approximately 10 megabase (Mb) conserved syntenic segment, the chromatin structures of the 2 homologous loci are strikingly different. The 2 previously unidentified hematopoietic cis-elements, one in each species, are not conserved in position and sequence and have enhancer activity in erythroid cells. In vivo, they both bind the transcription factors GATA1, SCL, LMO2, and Ldb1. More broadly, there are both species- and regulatory element–specific patterns of transcription factor binding. These findings suggest that some cis-elements regulating human and mouse GATA1 genes differ. More generally, mouse human sequence comparison may fail to identify all cis-elements.

A novel regulatory element between the human FGA and FGG genes

2012 ◽  
Vol 108 (09) ◽  
pp. 427-434 ◽  
Author(s):  
Richard J. Fish ◽  
Marguerite Neerman-Arbez
Keyword(s):  
Gene Cluster ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Luciferase Reporter ◽  
Transgenic Zebrafish ◽  
Regulatory Sequences ◽  
Gene Promoters ◽  
Cvd Risk ◽  
Enhancer Activity

SummaryHigh circulating fibrinogen levels correlate with cardiovascular disease (CVD) risk. Fibrinogen levels vary between people and also change in response to physiological and environmental stimuli. A modest proportion of the variation in fibrinogen levels can be explained by genotype, inferring that variation in genomic sequences that regulate the fibri-nogen genes (FGA, FGB and FGG) may affect hepatic fibrinogen production and perhaps CVD risk. We previously identified a conserved liver enhancer in the fibrinogen gene cluster (CNC12), between FGB and FGA. Genome-wide Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that transcription factors which bind fibrinogen gene promoters also interact with CNC12, as well as two potential fibrinogen enhancers (PFE), between FGA and FGG. Here we show that one of the PFE sequences has potent hepatocyte enhancer activity. Using a luciferase reporter gene system, we found that PFE2 enhances minimal promoter- and FGA promoter-driven gene expression in hepatoma cells, regardless of its orientation with respect to the promoters. A region within PFE2 bears a short series of conserved nucleotides which maintain enhancer activity without flanking sequence. We also demonstrate that PFE2 is a liver enhancer in vivo, driving enhanced green fluorescent protein expression in transgenic zebrafish larval livers. Our study shows that combining public domain ChIP-seq data with in vitro and in vivo functional tests can identify novel fibrinogen gene cluster regulatory sequences. Variation in such elements could affect fibrinogen production and influence CVD risk.

scholarly journals The Pu.1 Locus Is Differentially Regulated at the Level of Chromatin Structure and Noncoding Transcription by Alternate Mechanisms at Distinct Developmental Stages of Hematopoiesis

2007 ◽  
Vol 27 (21) ◽  
pp. 7425-7438 ◽  
Author(s):  
Maarten Hoogenkamp ◽  
Hanna Krysinska ◽  
Richard Ingram ◽  
Gang Huang ◽  
Rachael Barlow ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
B Cells ◽  
Regulatory Element ◽  
Precursor Cells ◽  
Hematopoietic Stem ◽  
Tissue Specific ◽  
Specific Regulation

ABSTRACT The Ets family transcription factor PU.1 is crucial for the regulation of hematopoietic development. Pu.1 is activated in hematopoietic stem cells and is expressed in mast cells, B cells, granulocytes, and macrophages but is switched off in T cells. Many of the transcription factors regulating Pu.1 have been identified, but little is known about how they organize Pu.1 chromatin in development. We analyzed the Pu.1 promoter and the upstream regulatory element (URE) using in vivo footprinting and chromatin immunoprecipitation assays. In B cells, Pu.1 was bound by a set of transcription factors different from that in myeloid cells and adopted alternative chromatin architectures. In T cells, Pu.1 chromatin at the URE was open and the same transcription factor binding sites were occupied as in B cells. The transcription factor RUNX1 was bound to the URE in precursor cells, but binding was down-regulated in maturing cells. In PU.1 knockout precursor cells, the Ets factor Fli-1 compensated for the lack of PU.1, and both proteins could occupy a subset of Pu.1 cis elements in PU.1-expressing cells. In addition, we identified novel URE-derived noncoding transcripts subject to tissue-specific regulation. Our results provide important insights into how overlapping, but different, sets of transcription factors program tissue-specific chromatin structures in the hematopoietic system.

scholarly journals A New Role for Sterol Regulatory Element Binding Protein 1 Transcription Factors in the Regulation of Muscle Mass and Muscle Cell Differentiation

2009 ◽  
Vol 30 (5) ◽  
pp. 1182-1198 ◽  
Author(s):  
Virginie Lecomte ◽  
Emmanuelle Meugnier ◽  
Vanessa Euthine ◽  
Christine Durand ◽  
Damien Freyssenet ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Muscle Mass ◽  
Target Genes ◽  
Binding Protein ◽  
Regulatory Element ◽  
Element Binding Protein ◽  
Sterol Regulatory Element ◽  
Myogenic Program

ABSTRACT The role of the transcription factors sterol regulatory element binding protein 1a (SREBP-1a) and SREBP-1c in the regulation of cholesterol and fatty acid metabolism has been well studied; however, little is known about their specific function in muscle. In the present study, analysis of recent microarray data from muscle cells overexpressing SREBP1 suggested that they may play a role in the regulation of myogenesis. We then demonstrated that SREBP-1a and -1c inhibit myoblast-to-myotube differentiation and also induce in vivo and in vitro muscle atrophy. Furthermore, we have identified the transcriptional repressors BHLHB2 and BHLHB3 as mediators of these effects of SREBP-1a and -1c in muscle. Both repressors are SREBP-1 target genes, and they affect the expression of numerous genes involved in the myogenic program. Our findings identify a new role for SREBP-1 transcription factors in muscle, thus linking the control of muscle mass to metabolic pathways.

scholarly journals Genome-wide analysis of cis-regulatory changes in the metabolic adaptation of cavefish

2020 ◽  
Author(s):  
Jaya Krishnan ◽  
Chris W. Seidel ◽  
Ning Zhang ◽  
Jake VanCampen ◽  
Robert Peuß ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Metabolic Adaptation ◽  
Enhancer Activity ◽  
Genome Wide ◽  
Cave Environment

AbstractChanges in cis-regulatory elements play important roles in adaptation and phenotypic evolution. However, their contribution to metabolic adaptation of organisms is less understood. Here we have utilized a unique vertebrate model, Astyanax mexicanus, different morphotypes of which survive in nutrient-rich surface and nutrient-deprived cave water to uncover gene regulatory networks in metabolic adaptation. We performed genome-wide epigenetic profiling in the liver tissue of one surface and two independently derived cave populations. We find that many cis-regulatory elements differ in their epigenetic status/chromatin accessibility between surface fish and cavefish, while the two independently derived cave populations have evolved remarkably similar regulatory signatures. These differentially accessible regions are associated with genes of key pathways related to lipid metabolism, circadian rhythm and immune system that are known to be altered in cavefish. Using in vitro and in vivo functional testing of the candidate cis-regulatory elements, we find that genetic changes within them cause quantitative expression differences. We characterized one cis-regulatory element in the hpdb gene and found a genomic deletion in cavefish that abolishes binding of the transcriptional repressor IRF2 in vitro and derepresses enhancer activity in reporter assays. Genetic experiments further validated a cis-mediated role of the enhancer and suggest a role of this deletion in the upregulation of hpdb in wild cavefish populations. Selection of this mutation in multiple independent cave populations supports its importance in the adaptation to the cave environment, providing novel molecular insights into the evolutionary trade-off between loss of pigmentation and adaptation to a food-deprived cave environment.

scholarly journals Parallel functional testing identifies enhancers active in early postnatal mouse brain

2021 ◽  
Author(s):  
Jason T. Lambert ◽  
Linda Su-Feher ◽  
Karol Cichewicz ◽  
Tracy L. Warren ◽  
Iva Zdilar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
Dna Sequences ◽  
Regulatory Element ◽  
Linkage Disequilibrium Block ◽  
Regulatory Elements ◽  
Functional Screening ◽  
Functional Testing ◽  
Enhancer Activity

ABSTRACTCis-regulatory elements such as enhancers play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted the self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identify putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in normal and pathogenic brain development.

scholarly journals A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface

2016 ◽  
Vol 113 (19) ◽  
pp. 5364-5369 ◽  
Author(s):  
Leonardo M. R. Ferreira ◽  
Torsten B. Meissner ◽  
Tarjei S. Mikkelsen ◽  
William Mallard ◽  
Charles W. O’Donnell ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Regulatory Element ◽  
Saturation Mutagenesis ◽  
Central Component ◽  
Immune Gene ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
A Cell ◽  
Maternal Fetal Interface

HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.

scholarly journals Cell-Type-Specific Activation and Repression of PU.1 by a Complex of Discrete, Functionally Specialized cis-Regulatory Elements

2010 ◽  
Vol 30 (20) ◽  
pp. 4922-4939 ◽  
Author(s):  
Mark A. Zarnegar ◽  
Jing Chen ◽  
Ellen V. Rothenberg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory Element ◽  
Cell Lineage ◽  
Myeloid Cell ◽  
Regulatory Elements ◽  
Cell Type ◽  
Enhancer Activity ◽  
Cell Type Specific

ABSTRACT The transcription factor PU.1 is critical for multiple hematopoietic lineages, but different leukocyte types require strictly distinct patterns of PU.1 regulation. PU.1 is required early for T-cell lineage development but then must be repressed by a stage-specific mechanism correlated with commitment. Other lineages require steady, low expression or upregulation. Until now, only the promoter plus a distal upstream regulatory element (URE) could be invoked to explain nearly all Sfpi1 (PU.1) activation and repression, including bifunctional effects of Runx1. However, the URE is dispensable for most Sfpi1 downregulation in early T cells, and we show that it retains enhancer activity in immature T-lineage cells even where endogenous Sfpi1 is repressed. We now present evidence for another complex of conserved noncoding elements that mediate discrete, cell-type-specific regulatory features of Sfpi1, including a myeloid cell-specific activating element and a separate, pro-T-cell-specific silencer element. These elements yield opposite, cell-type-specific responses to Runx1. T-cell-specific repression requires Runx1 acting through multiple nonconsensus sites in the silencer core. These newly characterized sites recruit Runx1 binding in early T cells in vivo and define a functionally specific scaffold for dose-dependent, Runx-mediated repression.

scholarly journals Transcriptional networks driving enhancer function in the CFTR gene

2012 ◽  
Vol 446 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Jenny L. Kerschner ◽  
Ann Harris
Keyword(s):  
Regulatory Element ◽  
Regulatory Elements ◽  
Transcriptional Networks ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Dnase I Footprinting ◽  
Gene Assay ◽  
Nuclear Factor 1

A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene.

Enhancer-dependent inhibition of mouse renin transcription by inflammatory cytokines

2005 ◽  
Vol 288 (1) ◽  
pp. F117-F124 ◽  
Author(s):  
Li Pan ◽  
Yanping Wang ◽  
Craig A. Jones ◽  
Sean T. Glenn ◽  
Heinz Baumann ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Inflammatory Cytokines ◽  
Mutational Analysis ◽  
Fold Increase ◽  
Oncostatin M ◽  
Flanking Sequence ◽  
Receptor Binding Site ◽  
Enhancer Activity

Inflammatory cytokines have been shown to inhibit renin gene expression in the kidney in vivo and the kidney tumor-derived As4.1 cell line. In this report, we show that cytokines oncostatin M (OSM), IL-6, and IL-1β inhibit transcriptional activity associated with 4.1 kb of the mouse renin 5′-flanking sequence in As4.1 cells. The 242-bp enhancer (−2866 to −2625 bp) is sufficient to mediate the observed inhibitory effects. Sequences within the enhancer required for inhibition by each of these cytokines have been determined by deletional and mutational analysis. Results indicate that a 39-bp region (CEC) containing a cAMP-responsive element, an E-box, and a steroid receptor-binding site, previously identified as the most critical elements for enhancer activity, is sufficient for the inhibition induced by IL-1β. However, mutation of each of the three component sites does not abolish the inhibition by IL-1β, suggesting that the target(s) of cytokine action may not be the transcription factors binding directly to these sites. This CEC region is also critical, but not sufficient, for the inhibition mediated by OSM and IL-6. These data suggest that the direct target of the associated cytokines may be coactivators interacting with transcription factors binding at the enhancer. Finally, we show that OSM treatment caused a 17-fold increase in promoter activity when only 2,625 bp of the Ren-1cflanking sequence were tested, in which the enhancer is not present. Three regions including −2625 to −1217 bp, the HOX·PBX binding site at −60 bp, and −59 to +6 bp have been found to contribute to this induction.

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