scholarly journals Lmx1a drives Cux2 expression in the cortical hem through activation of a conserved intronic enhancer

2018 ◽  
Author(s):  
Santiago P. Fregoso ◽  
Brett E. Dwyer ◽  
Santos J. Franco
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Expression Patterns ◽  
Neural Progenitors ◽  
Transcriptional Networks ◽  
Enhancer Activity ◽  
Homeobox Transcription Factor ◽  
Cortical Hem

AbstractDuring neocortical development, neurons are produced by a diverse pool of neural progenitors. A subset of progenitors express the Cux2 gene and are fate-restricted to produce certain neuronal subtypes, but the upstream pathways that specify these progenitor fates remain unknown. To uncover the transcriptional networks that regulate Cux2 expression in the forebrain, we characterized a conserved Cux2 enhancer that we find recapitulates Cux2 expression specifically in the cortical hem. Using a bioinformatic approach, we found several potential transcription factor (TF) binding sites for cortical hem-patterning TFs. We found that the homeobox transcription factor, Lmx1a, can activate the Cux2 enhancer in vitro. Furthermore, we show that multiple Lmx1a binding sites required for enhancer activity in the cortical hem in vivo. Mis-expression of Lmx1a in neocortical progenitors caused an increase in Cux2+-lineage cells. Finally, we compared several conserved human enhancers with cortical hem-restricted activity and found that recurrent Lmx1a binding sites are a top shared feature. Uncovering the network of TFs involved in regulating Cux2 expression will increase our understanding of the mechanisms pivotal in establishing Cux2-lineage fates in the developing forebrain.Summary StatementAnalysis of a cortical hem-specific Cux2 enhancer reveals role for Lmx1a as a critical upstream regulator of Cux2 expression patterns in neural progenitors during early forebrain development.

scholarly journals Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

2021 ◽  
Vol 49 (7) ◽  
pp. 3856-3875
Author(s):  
Marina Kulik ◽  
Melissa Bothe ◽  
Gözde Kibar ◽  
Alisa Fuchs ◽  
Stefanie Schöne ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Dna Binding ◽  
Receptor Binding ◽  
Binding Sites ◽  
Specific Gene ◽  
Functional Diversification ◽  
Enhancer Activity ◽  
Sequence Composition

Abstract The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

scholarly journals The homeodomain region controls the phenotype of HOX-induced murine leukemia

Blood ◽  
2012 ◽  
Vol 120 (19) ◽  
pp. 4018-4027 ◽  
Author(s):  
Constanze Breitinger ◽  
Emanuel Maethner ◽  
Maria-Paz Garcia-Cuellar ◽  
Robert K. Slany
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Expression Patterns ◽  
Hox Proteins ◽  
Hematopoietic Development ◽  
N Terminus ◽  
Fast Acting ◽  
Murine Leukemia

Abstract HOX proteins are widely involved in hematopoietic development. These transcription factors combine a conserved DNA-binding homeobox with a divergent N-terminus that mediates interaction with variable cofactors. The resulting combinatorial diversity is thought to be responsible for mammalian HOX specificity. Contrasting this proposed mechanism for normal HOX function, here we demonstrate that, in the context of hematopoietic immortalization and leukemogenesis, individual HOX properties are governed almost exclusively by the homeodomain. Swap experiments between HOXA1 and HOXA9, 2 members of nonrelated paralog groups, revealed that gene expression patterns of HOX transformed cells in vitro are determined by the nature of the homeodomain. Similar results were seen in vivo during HOX-mediated leukemogenesis. An exchange of the homeodomains was sufficient to convert the slow, low-penetrance phenotype of HOXA1-induced leukemia to the aggressive fast-acting disease elicited by HOXA9 and vice versa. Mutation and deletion studies identified several subregions within the DNA binding domain responsible for paralog specificity. Previously defined binding sites for PBX cofactors within the exchangeable, nonhomeobox segment were dispensable for in vitro oncogenic HOX activity but affected in vivo disease development. The transcriptional activator domain shared by HOXA1 and HOXA9 at the very N-terminus proved essential for all transformation.

scholarly journals Dynamic expression pattern and subcellular localization of the Rhox10 homeobox transcription factor during early germ cell development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 611-624 ◽  
Author(s):  
Hye-Won Song ◽  
Christina T Dann ◽  
John R McCarrey ◽  
Marvin L Meistrich ◽  
Gail A Cornwall ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Germ Cells ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Germline Stem Cells ◽  
Dynamic Expression ◽  
Homeobox Transcription Factor ◽  
Dramatic Shift

Homeobox genes encode transcription factors that regulate diverse developmental events. The largest known homeobox gene cluster – the X-linked mouse reproductive homeobox (Rhox) cluster – harbors genes whose expression patterns and functions are largely unknown. Here, we report that a member of this cluster, Rhox10, is expressed in male germ cells. Rhox10 is highly transcribed in spermatogonia in vivo and is upregulated in response to the differentiation-inducing agent retinoic acid in vitro. Using a specific RHOX10 antiserum that we generated, we found that RHOX10 protein is selectively expressed in fetal gonocytes, germline stem cells, spermatogonia, and early spermatocytes. RHOX10 protein undergoes a dramatic shift in subcellular localization as germ cells progress from mitotically arrested gonocytes to mitotic spermatogonia and from mitotic spermatogonia to early meiotic spermatocytes, consistent with RHOX10 performing different functions in these stages.

scholarly journals Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

2017 ◽  
Author(s):  
Luca Tosti ◽  
James Ashmore ◽  
Boon Siang Nicholas Tan ◽  
Benedetta Carbone ◽  
Tapan K Mistri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Dna Interactions

AbstractThe identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

beta3-tubulin is directly repressed by the engrailed protein in Drosophila

Development ◽  
1997 ◽  
Vol 124 (13) ◽  
pp. 2527-2536 ◽  
Author(s):  
N. Serrano ◽  
H.W. Brock ◽  
F. Maschat
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transgenic Line ◽  
Target Genes ◽  
Regulatory Protein ◽  
Polytene Chromosomes ◽  
First Intron ◽  
Direct Target

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the beta3-tubulin gene, as a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of beta3-tubulin is derepressed in the ectoderm of engrailed mutant embryos. Repression of beta3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis.

Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites

1990 ◽  
Vol 10 (4) ◽  
pp. 1432-1438
Author(s):  
D M Ruden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Schizosaccharomyces Pombe ◽  
Binding Sites ◽  
Transcription Activator ◽  
Integral Number ◽  
Dna Binding Site ◽  
Adh1 Gene

When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S. pombe in vivo by an endogenous transcription factor. In vitro studies show that this S. pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively. Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.

Synergistic activation of a human promoter in vivo by transcription factor Sp1

1991 ◽  
Vol 11 (4) ◽  
pp. 1935-1943
Author(s):  
G M Anderson ◽  
S O Freytag
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Single Copy ◽  
Relative Contribution ◽  
Human Promoter ◽  
Transcription Factor Sp1 ◽  
Synergistic Activation ◽  
Cellular Context

Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.

Abstract 408: Identifying the Molecular Basis of Monocyte Development Using Enhancer Profiling

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Graham D Thomas ◽  
Richard N Hanna ◽  
Christopher K Glass ◽  
Catherine C Hedrick
Keyword(s):  
Transcription Factor ◽  
Indirect Evidence ◽  
Hierarchical Cluster ◽  
Enhancer Activity ◽  
Vascular Integrity ◽  
Super Enhancer ◽  
A Genome ◽  
Primary Mouse

Intro: Monocytes (Mo) are key drivers of atherosclerosis. In mouse the two main monocyte populations are distinguished based on Ly6C expression. Classical Ly6Chi Mo drive atherosclerosis while Ly6Clow Mo help to maintain vascular integrity. Indirect evidence also suggests that Ly6Clow Mo protect against atherosclerosis. Mo develop in the bone marrow (BM) from the macrophage dendritic cell precursor (MDP) and common monocyte progenitor (cMoP), although precise ontological relationships are unclear. We have recently described an obligate cell-intrinsic role for the transcription factor Nr4a1 in Ly6Clow monocyte development. Hypothesis: Enhancers drive transcription factor-dependent programs of gene expression in a cell-specific manner. We hypothesize that a functional analysis of Nr4a1-associated enhancers will provide insight into mechanisms regulating Ly6Clow Mo development. Furthermore, a genome-wide analysis of Mo and their progenitor enhancers will consolidate our understanding of lineage relationships between these cells. Methods: Using H3K4me2 and H3K27ac to define enhancers and enhancer activity respectively we have performed ChIP-Seq on primary mouse MDP, cMoP, Ly6Chi and Ly6Clow Mo. Results: Of 114,755 enhancers 10,070 were differentially regulated. Hierarchical cluster analysis of these regions supports the consensus view that Ly6Clow Mo arise from Ly6Chi Mo. A 20kb ‘super-enhancer’ spanning Nr4a1 (Nr4a1se) is selectively induced in Ly6Clo Mo. We have begun to dissect Nr4a1se to identify regulators of Ly6Clow monocyte development. Three Nr4a1se regions have in vitro enhancer activity and show high H3K27ac in CD14dimCD16+ Mo, the proposed orthologue of mouse Ly6Clow Mo. This is evidence that Nr4a1 expression is conserved between humans and mice. Conclusion: We have identified candidate enhancers regulating Ly6Clow monocyte development. We are currently knocking out these regions using the CRISPR-Cas9 system to test their role in Ly6Clow Mo development in vivo. We aim to present preliminary data from these experiments at this meeting.

scholarly journals Properties of initiator-associated transcription mediated by GAL4-VP16.

1993 ◽  
Vol 13 (12) ◽  
pp. 7469-7475 ◽  
Author(s):  
C Chang ◽  
J D Gralla
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Dna Binding Sites ◽  
Terminal Deoxynucleotide Transferase ◽  
Initiator Element ◽  
High Level ◽  
Primary Activation ◽  
Initial Assembly

Transcription associated with a terminal deoxynucleotide transferase gene initiator element is shown to respond to the transcription factor GAL4-VP16 both in vivo and in vitro. High-level transcription requires both an intact initiator element and bound activator. Transcription from this initiator-directed promoter is synergistic in vivo in that five GAL4 DNA binding sites yield 36 times the expression of a single site. Promoters dominated by initiator and TATA elements respond similarly to several GAL4-based activators, including GAL4-Sp1, GAL4-CTF, GAL4(1-147), GAL4-p53, GAL4-C/EBP, and GAL4-ER(EF), as well as GAL4-VP16 and Sp1. These and other similarities suggest that primary activation of TATA- and initiator-dominated promoters occurs at common steps. Since the initial assembly steps do not appear to be common for the two promoter types, the results place interesting constraints on models for how activation occurs.

scholarly journals The VEGF-regulated transcription factor HLX controls the expression of guidance cues and negatively regulates sprouting of endothelial cells

Blood ◽  
2011 ◽  
Vol 117 (9) ◽  
pp. 2735-2744 ◽  
Author(s):  
Julia Testori ◽  
Bernhard Schweighofer ◽  
Iris Helfrich ◽  
Caterina Sturtzel ◽  
Karoline Lipnik ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Endothelial Cell Migration ◽  
Gene Repertoire ◽  
Gene Encoding ◽  
Hypoxic Conditions ◽  
Homeobox Transcription Factor ◽  
Endothelial Growth Factor

Abstract The HLX gene encoding a diverged homeobox transcription factor has been found to be up-regulated by vascular endothelial growth factor-A (VEGF-A) in endothelial cells. We have now investigated the gene repertoire induced by HLX and its potential biologic function. HLX strongly increased the transcripts for several repulsive cell-guidance proteins including UNC5B, plexin-A1, and semaphorin-3G. In addition, genes for transcriptional repressors such as HES-1 were up-regulated. In line with these findings, adenoviral overexpression of HLX inhibited endothelial cell migration, sprouting, and vessel formation in vitro and in vivo, whereas proliferation was unaffected. This inhibition of sprouting was caused to a significant part by HLX-mediated up-regulation of UNC5B as shown by short hairpin RNA (shRNA)–mediated down-modulation of the respective mRNA. VEGF-A stimulation of endothelial cells induced elevated levels of HLX over longer time periods resulting in especially high up-regulation of UNC5B mRNA as well as an increase in cells displaying UNC5B at their surface. However, induction of HLX was strongly reduced and UNC5B up-regulation completely abrogated when cells were exposed to hypoxic conditions. These data suggest that HLX may function to balance attractive with repulsive vessel guidance by up-regulating UNC5B and to down-modulate sprouting under normoxic conditions.

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