scholarly journals Functional Interaction between the CoactivatorDrosophila CREB-Binding Protein and ASH1, a Member of the Trithorax Group of Chromatin Modifiers

2000 ◽  
Vol 20 (24) ◽  
pp. 9317-9330 ◽  
Author(s):  
Frédéric Bantignies ◽  
Richard H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Chromatin Structure ◽  
Binding Protein ◽  
Polytene Chromosomes ◽  
Open Chromatin ◽  
Creb Binding Protein ◽  
Trithorax Group ◽  
Chromatin Modifiers

ABSTRACT CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, we have begun a genetic analysis of CBP in flies. We have overproduced Drosophila melanogaster CBP (dCBP) in a variety of cell types and obtained distinct adult phenotypes. We used an uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: thePKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of thetrithorax group (trxG) of chromatin modifiers. Using immunocolocalization, we showed that the ASH1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. Our results thus implicate a second class of chromatin-associated proteins in mediating dCBP function and imply that dCBP might be involved in the regulation of higher-order chromatin structure.

scholarly journals Activation of Utrophin Promoter by Heregulin via theets-related Transcription Factor Complex GA-binding Protein α/β

1999 ◽  
Vol 10 (6) ◽  
pp. 2075-2086 ◽  
Author(s):  
Tejvir S. Khurana ◽  
Alan G. Rosmarin ◽  
Jing Shang ◽  
Thomas O. B. Krag ◽  
Saumya Das ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Transcription Factors ◽  
Neuromuscular Junction ◽  
Muscle Cell ◽  
Cell Cultures ◽  
Binding Protein ◽  
Duchenne's Muscular Dystrophy ◽  
Ga Binding Protein

Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate thatets-related GA-binding protein α/β transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein α/β complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal–regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne’s muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.

scholarly journals Site-Specific Acetylation by p300 or CREB Binding Protein Regulates Erythroid Krüppel-Like Factor Transcriptional Activity via Its Interaction with the SWI-SNF Complex

2001 ◽  
Vol 21 (7) ◽  
pp. 2413-2422 ◽  
Author(s):  
Wenjun Zhang ◽  
Shilpa Kadam ◽  
Beverly M. Emerson ◽  
James J. Bieker
Keyword(s):  
Chromatin Structure ◽  
Binding Protein ◽  
Globin Gene ◽  
Critical Role ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Creb Binding Protein ◽  
Site Specific ◽  
Acetylation Status

ABSTRACT Recruitment of modifiers and remodelers to specific DNA sites within chromatin plays a critical role in controlling gene expression. The study of globin gene regulation provides a convergence point within which to address these issues in the context of tissue-specific and developmentally regulated expression. In this regard, erythroid Krüppel-like factor (EKLF) is critical. EKLF is a red cell-specific activator whose presence is crucial for establishment of the correct chromatin structure and high-level transcriptional induction of adult β-globin. We now find, by metabolic labeling-immunoprecipitation experiments, that EKLF is acetylated in the erythroid cell. EKLF residues acetylated by CREB binding protein (CBP) in vitro map to Lys-288 in its transactivation domain and Lys-302 in its zinc finger domain. Although site-specific DNA binding by EKLF is unaffected by the acetylation status of either of these lysines, directed mutagenesis of Lys-288 (but not Lys-302) decreases the ability of EKLF to transactivate the β-globin promoter in vivo and renders it unable to be superactivated by coexpressed p300 or CBP. In addition, the acetyltransferase function of CBP or p300 is required for superactivation of wild-type EKLF. Finally, acetylated EKLF has a higher affinity for the SWI-SNF chromatin remodeling complex and is a more potent transcriptional activator of chromatin-assembled templates in vitro. These results demonstrate that the acetylation status of EKLF is critical for its optimal activity and suggest a mechanism by which EKLF acts as an integrator of remodeling and transcriptional components to alter chromatin structure and induce adult β-globin expression within the β-like globin cluster.

Chromatin Structure and Control of β-Like Globin Gene Switching

2002 ◽  
Vol 227 (9) ◽  
pp. 683-700 ◽  
Author(s):  
Susanna Harju ◽  
Kellie J. McQueen ◽  
Kenneth R. Peterson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Globin Gene ◽  
Cis Acting ◽  
Gene Switching ◽  
Globin Gene Expression ◽  
Chromatin Modifiers

The human β-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional β-like globin genes, ε, Gγ, Aγ, δ, and β, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNasel-hypersensitive sites located 6-22 Kb upstream of the ε-globin gene. During ontogeny two switches occur in β-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic ε- to fetal γ-globin occurs at six weeks of gestation. The second switch from γ- to adult δ- and β-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals Adhesion Behaviour of Primary Human Osteoblasts and Fibroblasts on Polyether Ether Ketone Compared with Titanium under In Vitro Lipopolysaccharide Incubation

Materials ◽  
2019 ◽  
Vol 12 (17) ◽  
pp. 2739 ◽  
Author(s):  
Korbinian Benz ◽  
Andreas Schöbel ◽  
Marisa Dietz ◽  
Peter Maurer ◽  
Jochen Jackowski
Keyword(s):  
Gene Expression ◽  
Protein Level ◽  
Binding Protein ◽  
Sem Images ◽  
Human Osteoblasts ◽  
Polyether Ether Ketone ◽  
Primary Human Osteoblasts ◽  
Titanium Surfaces ◽  
Ether Ketone

The aim of this in vitro pilot study was to analyse the adhesion behaviour of human osteoblasts and fibroblasts on polyether ether ketone (PEEK) when compared with titanium surfaces in an inflammatory environment under lipopolysaccharide (LPS) incubation. Scanning electron microscopy (SEM) images of primary human osteoblasts/fibroblasts on titanium/PEEK samples were created. The gene expression of the LPS-binding protein (LBP) and the LPS receptor (toll-like receptor 4; TLR4) was measured by real-time polymerase chain reaction (PCR). Immunocytochemistry was used to obtain evidence for the distribution of LBP/TLR4 at the protein level of the extra-cellular-matrix-binding protein vinculin and the actin cytoskeleton. SEM images revealed that the osteoblasts and fibroblasts on the PEEK surfaces had adhesion characteristics comparable to those of titanium. The osteoblasts contracted under LPS incubation and a significantly increased LBP gene expression were detected. This was discernible at the protein level on all the materials. Whereas no increase of TLR4 was detected with regard to mRNA concentrations, a considerable increase in the antibody reaction was detected on all the materials. As is the case with titanium, the colonisation of human osteoblasts and fibroblasts on PEEK samples is possible under pro-inflammatory environmental conditions and the cellular inflammation behaviour towards PEEK is lower than that of titanium.

scholarly journals Activating Signal Cointegrator 2 Belongs to a Novel Steady-State Complex That Contains a Subset of Trithorax Group Proteins

2003 ◽  
Vol 23 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Young-Hwa Goo ◽  
Young Chang Sohn ◽  
Dae-Hwan Kim ◽  
Seung-Whan Kim ◽  
Min-Jung Kang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Steady State ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Retinoic Acid Receptor ◽  
Dependent Manner ◽  
Trithorax Group ◽  
Trithorax Group Proteins

ABSTRACT Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, α/β-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

scholarly journals CBP/β-Catenin/FOXM1 Is a Novel Therapeutic Target in Triple Negative Breast Cancer

Cancers ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 525 ◽  
Author(s):  
Alexander Ring ◽  
Cu Nguyen ◽  
Goar Smbatyan ◽  
Debu Tripathy ◽  
Min Yu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Drug Resistance ◽  
Triple Negative Breast Cancer ◽  
Therapeutic Target ◽  
Triple Negative ◽  
Creb Binding Protein ◽  
Novel Therapeutic

Background: Triple negative breast cancers (TNBCs) are an aggressive BC subtype, characterized by high rates of drug resistance and a high proportion of cancer stem cells (CSC). CSCs are thought to be responsible for tumor initiation and drug resistance. cAMP-response element-binding (CREB) binding protein (CREBBP or CBP) has been implicated in CSC biology and may provide a novel therapeutic target in TNBC. Methods: RNA Seq pre- and post treatment with the CBP-binding small molecule ICG-001 was used to characterize CBP-driven gene expression in TNBC cells. In vitro and in vivo TNBC models were used to determine the therapeutic effect of CBP inhibition via ICG-001. Tissue microarrays (TMAs) were used to investigate the potential of CBP and associated proteins as biomarkers in TNBC. Results: The CBP/ß-catenin/FOXM1 transcriptional complex drives gene expression in TNBC and is associated with increased CSC numbers, drug resistance and poor survival outcome. Targeting of CBP/β-catenin/FOXM1 with ICG-001 eliminated CSCs and sensitized TNBC tumors to chemotherapy. Immunohistochemistry of TMAs demonstrated a significant correlation between FOXM1 expression and TNBC subtype. Conclusion: CBP/β-catenin/FOXM1 transcriptional activity plays an important role in TNBC drug resistance and CSC phenotype. CBP/β-catenin/FOXM1 provides a molecular target for precision therapy in triple negative breast cancer and could form a rationale for potential clinical trials.

scholarly journals Integrase-Specific Enhancement and Suppression of Retroviral DNA Integration by Compacted Chromatin Structure In Vitro

2004 ◽  
Vol 78 (11) ◽  
pp. 5848-5855 ◽  
Author(s):  
Konstantin D. Taganov ◽  
Isabel Cuesta ◽  
René Daniel ◽  
Lisa Ann Cirillo ◽  
Richard A. Katz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Chromatin Structure ◽  
Site Selection ◽  
Integration Site ◽  
Histone H1 ◽  
Detectable Effect ◽  
Host Chromosome ◽  
Dna Integration ◽  
Hiv 1

ABSTRACT Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

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