Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and -independent mechanisms

Development ◽  
1999 ◽  
Vol 126 (2) ◽  
pp. 281-292 ◽  
Author(s):  
D.J. Epstein ◽  
A.P. McMahon ◽  
A.L. Joyner
Keyword(s):  
Gene Expression ◽  
Sonic Hedgehog ◽  
Binding Sites ◽  
Transcriptional Regulators ◽  
Regulatory Sequences ◽  
Reporter Expression ◽  
Enhancer Activity ◽  
Ventral Midline ◽  
Genomic Clones ◽  
Anteroposterior Axis

The axial midline mesoderm and the ventral midline of the neural tube, the floor plate, share the property of being a source of the secreted protein, Sonic hedgehog (Shh), which has the capacity to induce a variety of ventral cell types along the length of the mouse CNS. To gain insight into the mechanisms by which Shh transcription is initiated in these tissues, we set out to identify the cis-acting sequences regulating Shh gene expression. As an approach, we have tested genomic clones encompassing 35 kb of the Shh locus for their ability to direct a lacZ reporter gene to the temporally and spatially restricted confines of the Shh expression domains in transgenic mice. Three enhancers were identified that directed lacZ expression to distinct regions along the anteroposterior axis including the ventral midline of the spinal cord, hindbrain, rostral midbrain and caudal diencephalon, suggesting that multiple transcriptional regulators are required to initiate Shh gene expression within the CNS. In addition, regulatory sequences were also identified that directed reporter expression to the notochord, albeit, under limited circumstances. Sequence analysis of the genomic clones responsible for enhancer activity from a variety of organisms, including mouse, chicken and human, have identified highly conserved binding sites for the hepatocyte nuclear factor 3 (Hnf3) family of transcriptional regulators in some, but not all, of the enhancers. Moreover, the generation of mutations in the Hnf3-binding sites showed their requirement in certain, but not all, aspects of Shh reporter expression. Taken together, our results support the existence of Hnf3-dependent and -independent mechanisms in the direct activation of Shh transcription within the CNS and axial mesoderm.

Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY

Development ◽  
2002 ◽  
Vol 129 (16) ◽  
pp. 3887-3899 ◽  
Author(s):  
Jonathan Gilthorpe ◽  
Marie Vandromme ◽  
Tim Brend ◽  
Alejandro Gutman ◽  
Dennis Summerbell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Specific Interaction ◽  
Transcriptional Regulators ◽  
Heterologous Promoter ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Hox Gene ◽  
Two Factors ◽  
Enhancer Function

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.

Target sequences for hunchback in a control region conferring Ultrabithorax expression boundaries

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1171-1179 ◽  
Author(s):  
C.C. Zhang ◽  
J. Muller ◽  
M. Hoch ◽  
H. Jackle ◽  
M. Bienz
Keyword(s):  
Long Range ◽  
Control Region ◽  
Binding Sites ◽  
Regulatory Sequences ◽  
Stripe Pattern ◽  
Anteroposterior Axis ◽  
Target Sequences ◽  
Beta Galactosidase ◽  
Control Regions

Boundaries of Ultrabithorax expression are mediated by long-range repression acting through the PBX or ABX control region. We show here that either of these control regions confers an early band of beta-galactosidase expression which is restricted along the anteroposterior axis of the blastoderm embryo. This band is succeeded by a stripe pattern with very similar anteroposterior limits. Dissection of the PBX control region demonstrates that the two patterns are conferred by distinct cis-regulatory sequences contained within separate PBX subfragments. We find several binding sites for hunchback protein within both PBX subfragments. Zygotic hunchback function is required to prevent ectopic PBX expression. Moreover, the PBX pattern is completely suppressed in embryos containing uniformly distributed maternal hunchback protein. Our results strongly suggest that hunchback protein directly binds to the PBX control region and acts as a repressor to specify the boundary positions of the PBX pattern.

scholarly journals Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

2019 ◽  
Vol 116 (20) ◽  
pp. 9893-9902 ◽  
Author(s):  
Christopher M. Uyehara ◽  
Daniel J. McKay
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Nuclear Receptor ◽  
Binding Sites ◽  
Transcriptional Responses ◽  
Enhancer Activity ◽  
Developmental Transitions ◽  
Experimental Systems ◽  
The Impact

The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.

scholarly journals NF-κB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-κB site in the ICAM-1 gene

2009 ◽  
Vol 38 (1) ◽  
pp. 42-53 ◽  
Author(s):  
Jiaping Xue ◽  
Prabhakar B. Thippegowda ◽  
Guochang Hu ◽  
Kurt Bachmaier ◽  
John W. Christman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Leukocyte Adhesion ◽  
Reporter Expression ◽  
Mobility Shift ◽  
Intron 1 ◽  
Tnf Α ◽  
Protease Activated Receptor 1

Activation of NF-κB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-κB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-κB binding sites in intron-1 (+70, NF-κB site 1; +611, NF-κB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-κB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-κB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (−1,385 to +234) construct ∼25-fold and mutation of intronic NF-κB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-α-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-κB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.

scholarly journals Evolution of lineage-specific functions in ancient cis -regulatory modules

Open Biology ◽  
2015 ◽  
Vol 5 (11) ◽  
pp. 150079 ◽  
Author(s):  
Stefan Pauls ◽  
Debbie K. Goode ◽  
Libero Petrone ◽  
Paola Oliveri ◽  
Greg Elgar
Keyword(s):  
Gene Expression ◽  
Morphological Evolution ◽  
Regulatory Sequences ◽  
Enhancer Activity ◽  
Regulatory Modules ◽  
History Of ◽  
Flanking Region ◽  
Regulatory Functions ◽  
Conserved Core ◽  
Flanking Regions

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis -regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.

scholarly journals Accurate and sensitive quantification of protein-DNA binding affinity

2018 ◽  
Vol 115 (16) ◽  
pp. E3692-E3701 ◽  
Author(s):  
Chaitanya Rastogi ◽  
H. Tomas Rube ◽  
Judith F. Kribelbauer ◽  
Justin Crocker ◽  
Ryan E. Loker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Biophysical Model ◽  
Regulatory Sequences ◽  
Binding Modes ◽  
Perfect Agreement ◽  
Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

scholarly journals Molecular competition can shape enhancer activity in the Drosophila embryo

2021 ◽  
Author(s):  
Rachel Waymack ◽  
Mario Gad ◽  
Zeba Wunderlich
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Drosophila Embryo ◽  
Enhancer Activity ◽  
Endogenous Gene ◽  
Endogenous Genes ◽  
Regulatory Dna ◽  
The Impact

Transgenic reporters allow the measurement of regulatory DNA activity in vivo and consequently have long been useful tools in the study of enhancers. Despite the utility of transgenic reporters, few studies have investigated the potential effects these reporters have on the expression of other transgenic reporters or endogenous genes. A full understanding of the impacts transgenic reporters have on expression is required for accurate interpretation of transgenic reporter data and characterization of gene regulatory mechanisms. Here, we investigate the impact transgenic reporters have on the expression of other transgenic reporters and endogenous genes. By measuring the expression of Kruppel (Kr) enhancer reporters in live Drosophila embryos that contain either one or two copies of identical reporters, we find reporters have an inhibitory effect on one another's expression. Further, expression of a nearby endogenous gene is decreased in the presence of a Kr enhancer reporter. Through the use of competitor binding site arrays, we present evidence that reporters, and potentially endogenous genes, are competing for transcription factors (TFs). Increasing the number of competitor Bcd binding sites decreases the peak levels and spatial extent of Bcd-regulated enhancer reporters' expression. To understand how small numbers of added TF binding sites could impact gene expression to the extent we observe, we develop a simple thermodynamic model of our system. Our model predicts competition of the measured magnitude specifically if TF binding is restricted to distinct nuclear subregions, underlining the importance of the non-homogenous nature of the nucleus in regulating gene expression.

scholarly journals Positional specificity of different transcription factor classes within enhancers

2018 ◽  
Vol 115 (30) ◽  
pp. E7222-E7230 ◽  
Author(s):  
Sharon R. Grossman ◽  
Jesse Engreitz ◽  
John P. Ray ◽  
Tung H. Nguyen ◽  
Nir Hacohen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Cell Types ◽  
Regulatory Sequences ◽  
Functional Characteristics ◽  
Positional Specificity ◽  
Chromatin Remodelers ◽  
Positional Bias

Gene expression is controlled by sequence-specific transcription factors (TFs), which bind to regulatory sequences in DNA. TF binding occurs in nucleosome-depleted regions of DNA (NDRs), which generally encompass regions with lengths similar to those protected by nucleosomes. However, less is known about where within these regions specific TFs tend to be found. Here, we characterize the positional bias of inferred binding sites for 103 TFs within ∼500,000 NDRs across 47 cell types. We find that distinct classes of TFs display different binding preferences: Some tend to have binding sites toward the edges, some toward the center, and some at other positions within the NDR. These patterns are highly consistent across cell types, suggesting that they may reflect TF-specific intrinsic structural or functional characteristics. In particular, TF classes with binding sites at NDR edges are enriched for those known to interact with histones and chromatin remodelers, whereas TFs with central enrichment interact with other TFs and cofactors such as p300. Our results suggest distinct regiospecific binding patterns and functions of TF classes within enhancers.

scholarly journals The enhancer activity of long interspersed nuclear element derived microRNA 625 induced by NF-κB

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hee-Eun Lee ◽  
Sang-Je Park ◽  
Jae-Won Huh ◽  
Hiroo Imai ◽  
Heui-Soo Kim
Keyword(s):  
Gene Expression ◽  
Transposable Elements ◽  
Human Genome ◽  
Dna Sequences ◽  
Binding Sites ◽  
Luciferase Assay ◽  
Enhancer Activity ◽  
Long Interspersed Nuclear Elements ◽  
Long Interspersed Nuclear Element ◽  
Affect Gene Expression

AbstractTransposable elements (TEs) are DNA sequences that cut or introduced into the genome, and they represent a massive portion of the human genome. TEs generate a considerable number of microRNAs (miRNAs) are derived from TEs (MDTEs). Numerous miRNAs are related to cancer, and hsa-miRNA-625 is a well-known oncomiR derived from long interspersed nuclear elements (LINEs). The relative expression of hsa-miRNA-625-5p differs in humans, chimpanzees, crab-eating monkeys, and mice, and four primers were designed against the 3′UTR of GATAD2B to analyze the different quantities of canonical binding sites and the location of miRNA binding sites. Luciferase assay was performed to score for the interaction between hsa-miRNA-625 and the 3′UTR of GATAD2B, while blocking NF-κB. In summary, the different numbers of canonical binding sites and the locations of miRNA binding sites affect gene expression, and NF-κB induces the enhancer activity of hsa-miRNA-625-5p by sharing the binding sites.

scholarly journals The LysR-Type Nitrogen Assimilation Control Protein Forms Complexes with Both Long and Short DNA Binding Sites in the Absence of Coeffectors

2010 ◽  
Vol 192 (19) ◽  
pp. 4827-4833 ◽  
Author(s):  
Christopher J. Rosario ◽  
Ryan L. Frisch ◽  
Robert A. Bender
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Binding Sites ◽  
Nitrogen Assimilation ◽  
Dna Bending ◽  
Transcriptional Regulators ◽  
The Other ◽  
Dna Binding Sites ◽  
Control Protein ◽  
Half Site

ABSTRACT Most LysR-type transcriptional regulators (LTTRs) function as tetramers when regulating gene expression. The nitrogen assimilation control protein (NAC) generally functions as a dimer when binding to DNA and activating transcription. However, at some sites, NAC binds as a tetramer. Like many LTTRs, NAC tetramers can recognize sites with long footprints (74 bp for the site at nac) with a substantial DNA bend or short footprints (56 bp for the site at cod) with less DNA bending. However, unlike other LTTRs, NAC can recognize both types of sites in the absence of physiologically relevant coeffectors, suggesting that the two conformers of the NAC tetramer (extended and compact) are interchangeable without the need for any modification to induce or stabilize the change. In order for NAC to bind as a tetramer, three interactions must exist: an interaction between the two NAC dimers and an interaction between each NAC dimer and its corresponding binding site. The interaction between one dimer and its DNA site can be weak (recognizing a half-site rather than a full dimer-binding site), but the other two interactions must be strong. Since the conformation of the NAC tetramer (extended or compact) is determined by the nature of the DNA site without the intervention of a small molecule, we argue that the coeffector that determines the conformation of the NAC tetramer is the DNA site to which it binds.

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